4.2 Electrophysiological characterization of single AME

Transcrição

Physiological analysis of central and peripheral
insect circadian pacemaker neurons:
Accessory medulla neurons of the Madeira cockroach
Rhyparobia maderae and olfactory receptor neurons of the
hawkmoth Manduca sexta
Dissertation
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Nico Werner Funk
Universität Kassel – Fachbereich 10
Mathematik und Naturwissenschaften
Institut für Biologie – Abteilung Tierphysiologie
Kassel, Juni 2015
II
Vom Fachbereich 10 Mathematik und Naturwissenschaften der Universität Kassel als
Dissertation am 19.06.2015 angenommen.
1. Gutachterin:
2. Gutachter:
Prof. Dr. Monika Stengl
PD Dr. Dieter Wicher
Prüfungskommission:
1. Prof. Dr. Monika Stengl
(Tierphysiologie, Universität Kassel)
2. PD Dr. Dieter Wicher
(Evolutionäre Neuroethologie, Max Planck Institut für chemische Ökologie, Jena)
3. Prof. Dr. Mireille A. Schäfer
(Zoologie - Schwerpunkt Entwicklungsbiologie, Universität Kassel)
4. Prof. Dr. Jörg H. Kleinschmidt
(Biophysik, Universität Kassel)
Tag der mündlichen Prüfung: 07.09.2015
III
IV
Eidesstattliche Erklärung
Hiermit versichere ich, dass ich die vorliegende Dissertation selbstständig, ohne unerlaubte
Hilfe Dritter angefertigt und andere als die in der Dissertation angegebenen Hilfsmittel nicht
benutzt habe. Alle Stellen, die wörtlich oder sinngemäß aus veröffentlichten oder
unveröffentlichten Schriften entnommen sind, habe ich als solche kenntlich gemacht. Dritte
waren an der inhaltlich-materiellen Erstellung der Dissertation nicht beteiligt; insbesondere
habe ich hierfür nicht die Hilfe eines Promotionsberaters in Anspruch genommen. Kein Teil
dieser Arbeit ist in einem anderen Promotions- oder Habilitationsverfahren verwendet
worden.
Kassel, 18.06.2015
Nico Funk
V
VI
„So eine Arbeit wird eigentlich nie fertig,
man muss sie für fertig erklären,
wenn man nach Zeit und Umständen
das Möglichste getan hat.“
Johann Wolfgang von Goethe
(Deutscher Dichter, 1749 - 1832)
VII
VIII
Contents
Contents
Contents _________________________________________________________________ IX
Contribution statement _____________________________________________________XV
List of abbreviations ______________________________________________________ XVII
Zusammenfassung _______________________________________________________ XXV
Summary ______________________________________________________________ XXVII
1
Introduction ______________________________________________________ 1
1.1
A short introduction into chronobiology_____________________________________ 1
1.2
General characteristics of circadian clocks ___________________________________ 2
1.3
The central circadian clock of the fruit fly Drosophila melanogaster ______________ 4
1.3.1 Molecular rhythms are generated by transcriptional-translational feedback regulation ________ 4
1.3.2 Molecular mechanisms underlying light entrainment ____________________________________ 8
1.3.3 Clock neurons of D. melanogaster ___________________________________________________ 9
1.3.3.1
Clock neurons of D. melanogaster innervate the AME and the dorsal brain _____________ 10
1.3.4 Control of rhythmic locomotor activity ______________________________________________ 11
1.3.4.1
Fruit flies show activity peaks at dusk and dawn __________________________________ 11
1.3.4.2
Electrical activity plays a crucial role in the circadian system ________________________ 12
1.3.4.3
Properties of the ventrolateral neurons _________________________________________ 13
1.3.4.4
Rhythmicity is driven by a flexible network of different clock neurons _________________ 14
1.3.5 The function of the neuropeptide PDF _______________________________________________ 14
1.3.5.1
PDF is expressed in the ventrolateral clock neurons _______________________________ 14
1.3.5.2
About 60 % of the clock neurons express the PDF receptor _________________________ 16
1.3.5.3
The PDF receptor couples to Gαs and probably also to Gαq___________________________ 16
1.3.5.4
PDF signaling is required for robust molecular cycling and synchrony _________________ 18
1.3.5.5
Additional functions of PDF ___________________________________________________ 19
1.3.5.6
Parallels between PDF and VIP signaling _________________________________________ 19
1.4
The central circadian clock of the cockroach Rhyparobia maderae_______________ 20
1.4.1 The compound eyes are required for entrainment _____________________________________ 20
1.4.2 Localization of the pacemaker in the optic lobes_______________________________________ 21
1.4.3 The bilateral pacemakers are mutually coupled _______________________________________ 23
1.4.4 Cellular identity of the central pacemaker ____________________________________________ 23
1.4.4.1
The accessory medulla and associated PDF-ir neurons _____________________________ 23
1.4.4.2
Transplantation studies located the pacemaker to the AME _________________________ 25
1.4.4.3
The AME is composed of glomeruli _____________________________________________ 25
1.4.5 Neurons associated with the accessory medulla _______________________________________ 26
1.4.6 Medulla cell groups involved in bilateral coupling ______________________________________ 27
1.4.7 Neurotransmitters of the accessory medulla __________________________________________ 27
1.4.7.1
Pigment-dispersing factor in the AME ___________________________________________ 28
1.4.7.2
Other neuropeptides and transmitters of the AME ________________________________ 32
1.4.8 Clock genes ____________________________________________________________________ 37
1.4.9 Model of the AME _______________________________________________________________ 38
1.4.10 Physiological characterization of the accessory medulla neurons _________________________ 39
1.4.10.1 AME neurons are coupled to synchronous spiking assemblies _______________________ 39
IX
Contents
1.4.10.2
1.4.10.3
1.4.10.4
1.5
PDF synchronizes AME neurons________________________________________________ 40
AME neurons show a predominant period length of 2 hours ________________________ 40
Ion channels involved in the generation of spontaneous activity _____________________ 41
Peripheral circadian clocks ______________________________________________ 42
1.5.1 Mammalian peripheral clocks ______________________________________________________ 42
1.5.2 Peripheral clocks in insects ________________________________________________________ 42
1.5.2.1
Peripheral clocks of D. melanogaster ___________________________________________ 42
1.5.2.2
Differences between the central and peripheral molecular clocks ____________________ 43
1.5.2.3
Peripheral clocks in other insects than D. melanogaster ____________________________ 43
1.5.3 The antennal clock of insects ______________________________________________________ 44
1.5.3.1
Antennae, the insects' nose ___________________________________________________ 44
1.5.3.2
Odorants are sensed with different types of receptors _____________________________ 46
1.5.3.3
The coreceptor ORCO is required for membrane insertion of conventional ORs _________ 47
1.5.3.4
OR/ORCO complexes function as ion channels in vitro _____________________________ 49
1.5.3.5
ORCO as well as conventional ORs regulate spontaneous activity ____________________ 51
1.5.3.6
Odorant-induced metabotropic signaling cascades ________________________________ 52
1.5.3.7
Temporal control of olfactory sensitivity ________________________________________ 55
1.5.3.7.1 In D. melanogaster the ORNs are the pacemakers controlling olfactory sensitivity ____ 55
1.5.3.7.2 Similar to the ORNs, the GRNs control gustatory sensitivity in D. melanogaster ______ 56
1.5.3.7.3 In moths and cockroaches the ORNs also appear to be pacemakers ________________ 56
1.5.3.8
Pheromones and general odorants are Zeitgeber for entrainment ____________________ 57
1.5.3.9
Phase-discrepancies between olfactory sensitivity, mating, and locomotor activity ______ 58
1.6
2
Aim of this thesis ______________________________________________________ 58
Material and methods _____________________________________________ 61
2.1
2.1.1
2.1.2
Animal rearing ________________________________________________________ 61
Cockroach rearing _______________________________________________________________ 61
Moth rearing ___________________________________________________________________ 62
2.2
Cloning of M. sexta or- and snmp-1 genes __________________________________ 63
2.3
Cell culture ___________________________________________________________ 63
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
2.3.6
2.3.7
Preparation of culture dishes ______________________________________________________ 63
Preparation of cell culture media ___________________________________________________ 64
Primary AME cell cultures _________________________________________________________ 64
Primary M. sexta ORN cell cultures _________________________________________________ 67
M. sexta MRRL-CH1 cell culture ____________________________________________________ 67
HEK 293 cell culture______________________________________________________________ 68
SF9 cell culture _________________________________________________________________ 69
2.4
Immunocytochemistry __________________________________________________ 69
2.5
Electrophysiology ______________________________________________________ 71
2.5.1
2.5.2
2.6
2.6.1
2.6.2
2.7
2.7.1
Extracellular recordings from the isolated AME _______________________________________ 71
Patch clamp recordings of AME cells ________________________________________________ 72
Calcium Imaging _______________________________________________________ 74
Calcium imaging experiments on heterologous expression systems _______________________ 74
Calcium imaging experiments on M. sexta ORNs_______________________________________ 76
Data analysis__________________________________________________________ 77
Immunocytochemical analysis of heterologous M. sexta ORCO expression _________________ 77
X
Contents
2.7.2
2.7.3
2.7.4
2.7.5
2.7.6
2.8
3
Analysis of extracellularly recorded electrical activity of AME neurons _____________________ 78
Analysis of whole-cell patch clamp recordings of single AME neurons _____________________ 81
Analysis of calcium imaging data of heterologous expression systems _____________________ 82
Analysis of M. sexta ORN calcium imaging data _______________________________________ 83
Statistical analysis _______________________________________________________________ 84
Preparation of figures __________________________________________________ 84
Results _________________________________________________________ 85
3.1
3.1.1
3.1.2
3.1.3
3.1.4
3.1.5
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.3
Electrophysiological characterization of the R. maderae central clock network ____ 85
Network activity of isolated AME neurons ____________________________________________ 85
Glutamate inhibits AME neurons ___________________________________________________ 92
Different effects of PDF on neurons of the isolated AME ________________________________ 95
8-Br-cAMP but not 8-Br-cGMP mimics all classes of PDF effects __________________________ 98
EPAC might be involved in cAMP-dependent inhibitions of AME neurons __________________ 103
Electrophysiological characterization of single AME neurons __________________ 105
Primary AME cell cultures ________________________________________________________ 105
Whole-cell patch clamp recordings of AME neurons reveal different current components ____ 106
Development of the whole-cell current components during a recording __________________ 113
Pharmacological characterization of the whole-cell currents ____________________________ 116
Effects of PDF on different current components of AME neurons ________________________ 130
Most AME neurons in primary cell cultures are silent __________________________________ 136
Characterization of peripheral pacemaker neurons of M. sexta's antennal clock __ 141
3.3.1 Heterologous expression of olfactory receptors of M. sexta ____________________________ 141
3.3.1.1
Immunocytochemical characterization of heterologously expressed MsexORCO _______ 141
3.3.1.2
Basic calcium imaging experiments on HEK 293 cells ______________________________ 144
3.3.1.3
Deorphanization of the pheromone receptor candidates MsexOR-1 and MsexOR-4 _____ 149
3.3.1.4
Modulation of heterologously expressed MsexORCO _____________________________ 156
3.3.2 Characterization of M. sexta ORNs in primary cell cultures _____________________________ 163
3.3.2.1
Fura-2 AM loading of M. sexta ORNs___________________________________________ 163
3.3.2.2
Modulation of ORCO in primary M. sexta ORN cell cultures ________________________ 164
4
Discussion ______________________________________________________ 171
4.1
Network analysis of central R. maderae pacemaker neurons __________________ 171
4.1.1 Network properties of the isolated AME ____________________________________________ 171
4.1.1.1
The AME network mainly contains inhibitory synaptic interactions __________________ 171
4.1.1.2
Excitatory synaptic interactions in the AME _____________________________________ 172
4.1.1.3
Bursting and oscillations are a characteristic activity pattern of the isolated AME ______ 172
4.1.2 Glutamate contributes to inhibitory synaptic interactions of the AME ____________________ 173
4.1.2.1
Glutamate-dependent inhibitions appeared to be PTX-insensitive ___________________ 173
4.1.3 Different effects of PDF on the electrical activity of AME neurons ________________________ 174
4.1.4 Involvement of cyclic nucleotides in PDF signaling ____________________________________ 175
4.1.4.1
Effects of the cyclic nucleotide analogues were less frequent than PDF-effects_________ 175
4.1.4.2
cGMP signaling in the AME __________________________________________________ 176
4.1.4.3
PDF effects are mediated by cAMP but not cGMP ________________________________ 176
4.1.4.4
EPAC-signaling in AME neurons _______________________________________________ 177
4.2
4.2.1
4.2.2
Electrophysiological characterization of single AME neurons in primary cell cultures 178
Primary cell cultures of AME neurons ______________________________________________ 178
AME neurons express different whole-cell current components _________________________ 180
XI
Contents
4.2.3
4.2.4
4.2.5
4.2.6
4.2.7
4.3
Ion channels underlying IK and Iin,sust ________________________________________________ 181
All current components spontaneously decrease during whole-cell recordings _____________ 182
Pharmacological characterization of the current components ___________________________ 184
Most neurons in AME cell cultures do not spike ______________________________________ 187
Effects of PDF on single AME neurons ______________________________________________ 189
Analysis of peripheral pacemaker neurons in M. sexta antennae _______________ 194
4.3.1 Transiently transfected HEK 293 cells poorly expressed ORCO ___________________________ 194
4.3.2 Transiently transfected HEK 293 cells did not reliably respond to pheromone stimulation ____ 196
4.3.2.1
CaM-dependent desensitization appears unlikely ________________________________ 197
4.3.2.2
Replacement of MsexORCO by other ORCO orthologues or Gα15 did not improve the
response rate to bombykal __________________________________________________ 197
4.3.2.3
Coexpression of SNMP-1 did not specifically improve the bombykal responsiveness ____ 198
4.3.2.4
BSA as solvent did not improve the response rate to bombykal but caused OR/ORCO2+
independent [Ca ] increases ________________________________________________ 199
4.3.2.5
The response rate to C-15 was very low ________________________________________ 200
4.3.2.6
Apparently, the low response rate to pheromone stimulation was due to the low expression
of MsexORCO, MsexOR-1, and MsexOR-4 ______________________________________ 201
4.3.3 The majority of transiently transfected HEK 293 cells did not respond to ORCO modulation ___ 202
2+
4.3.3.1
MsexORCO appears to function as Ca permeable ion channel, which is activated by VUAA1
________________________________________________________________________ 202
4.3.3.2
Effects of coexpressed ORs on the VUAA1 sensitivity _____________________________ 203
2+
4.3.3.3
MsexORCO mediated spontaneous [Ca ] increases ______________________________ 203
4.3.3.4
MsexORCO appears to be activated by cAMP____________________________________ 204
2+
4.3.3.5
The amiloride derivatives HMA and MIA caused OR/ORCO-independent [Ca ] increases in
HEK 293 cells _____________________________________________________________ 204
4.3.3.6
The insect repellent DEET does not appear to block MsexORCO _____________________ 205
4.3.3.7
No inhibitory effect of the cation channel blocker ruthenium red was observed ________ 205
4.3.4 The loading of M. sexta ORNs with fura-2 AM was not affected by multidrug resistance
transporter blockers in vitro ______________________________________________________ 206
4.3.5 Primary M. sexta ORN cell cultures were not affected by ORCO modulation _______________ 207
4.3.5.1
ORCO agonists did not significantly activate M. sexta ORNs in vitro __________________ 207
4.3.5.2
The ORCO antagonist OLC15 did not affect M. sexta ORNs in calcium imaging experiments
________________________________________________________________________ 208
4.3.5.3
The effects of the amiloride derivatives HMA and MIA appeared to be ORCO-independent in
all cell types tested ________________________________________________________ 208
4.3.5.4
A characterization of ORCO in M. sexta ORNs was not accomplished _________________ 209
4.3.6 The role of ORCO in M. sexta pheromone transduction ________________________________ 210
4.3.6.1
Ionotropic versus metabotropic transduction mechanisms in insect olfaction __________ 210
4.3.6.2
Extracellular tip recordings did not support ionotropic pheromone transduction _______ 212
4.3.6.3
Does ORCO function as pacemaker channel? ____________________________________ 214
4.4
5
A comparison between central AME and peripheral ORN pacemaker neurons ____ 215
Appendix ______________________________________________________ 217
5.1
Manduca sexta rearing ________________________________________________ 217
5.2
Primer sequences _____________________________________________________ 219
5.3
Composition of solutions _______________________________________________ 219
5.3.1
5.3.2
Solutions for primary AME cell cultures _____________________________________________ 219
Solutions for primary M. sexta ORN cell cultures and the MRRL-CH1 cell line _______________ 221
XII
Contents
5.3.3
5.3.4
5.3.5
Solutions for HEK 293 cell culture __________________________________________________ 221
Solutions for immunocytochemistry _______________________________________________ 222
Solutions for electrophysiology and calcium imaging __________________________________ 223
5.4
Electrophysiological characterization of AME neurons at the network level ______ 224
5.5
Patch clamp analysis of single AME neurons _______________________________ 227
5.6
Immunocytochemical characterization of heterologously expressed MsexORCO __ 231
5.7
Calcium imaging experiments on heterologous expression systems _____________ 232
5.8
Calcium imaging experiments on primary M. sexta ORN cell cultures ___________ 238
6
Bibliography ____________________________________________________ 241
7
Acknowledgements ______________________________________________ 279
XIII
XIV
Contribution statement
The contribution of the author for each part of this thesis will be clearly stated as follows. Parts of the
thesis have already been published and exact wording is marked with quotation marks.
Chapter 3.1: Electrophysiological characterization of the R. maderae central
clock network
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Designed the experiments.
Conducted the majority of experiments. Some experiments were conducted under the
author's guidance in practical courses: Investigation of glutamate effects (3.1.2, Fig. 44, Fig.
45, Fig. 46) was performed together with Anastasia Pyanova and Simone Achenbach,
investigation of EPAC-specific cAMP effects (3.1.5, Fig. 56) together with Janis Sebastian
Brusius.
Analyzed the data, re-analyzed recordings from previous work (Funk 2005), (Fig. 47, Fig. 49,
Fig. 50, Fig. 55A, Fig. 122).
Prepared all figures and tables.
Wrote the manuscript.
Chapter 3.2: Electrophysiological characterization of single AME neurons
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Designed the first experimental series (6 mM CaCl2). The second experimental series (1 mM
CaCl2) was designed together with Hanzey Yasar.
Conducted the first experimental series. Primary cell cultures for the second experimental
series were prepared by Christa Uthof and patch clamp experiments were conducted by
Hanzey Yasar under the author's guidance.
Analyzed the data of the first experimental series, re-analyzed the raw data of the second
experimental series. The data shown in Fig. 68, Fig. 69, Fig. 77, Fig. 78, Fig. 79, and Fig. 126
were re-analyzed from (Yasar 2013).
A part of the results (PDF-dependent inhibitions of potassium and sodium currents) was
published in: Wei H, Yasar H, Funk NW, Giese M, Baz el S, Stengl M (2014) Signaling of
pigment-dispersing factor (PDF) in the Madeira cockroach Rhyparobia maderae. PLoS One 9
(9):e108757
A similar figure like Fig. 126 J appeared in (Wei et al. 2014). Fig. 117 was modified after (Wei
et al. 2014).
XV
Chapter 3.3: Characterization of peripheral pacemaker neurons of M. sexta's
antennal clock
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Designed all experiments.
Conducted the experiments. A minor part of the calcium imaging experiments was
performed by Dr. Latha Mukunda (Max Planck Institute for Chemical Ecology, Jena) under the
author's guidance: investigation of spontaneous and VUAA1-dependent [Ca2+] increases in
seven non-transfected HEK 293 cell cultures.
Cloning of M. sexta ORs, ORCO, and SNMP-1 was performed by Dr. Ewald Große-Wilde and
Sascha Bucks (Max Planck Institute for Chemical Ecology, Jena); cloning of D. melanogaster
SNMP-1 was performed by Jackson Sparks and Prof. Dr. Richard G. Vogt (University of South
Carolina, USA) and Dr. Jing-Jiang Zhou (Rothamsted Research, Harpenden, UK).
All HEK 293 and SF9 cell cultures were provided by Sabine Kaltofen or Sylke Dietel-Gläßer;
transient transfections were performed by Sabine Kaltofen or Sylke Dietel-Gläßer; all primary
M. sexta ORN cell cultures were prepared by Hongying Wei, El-Sayed Baz, or Christa Uthof.
Analyzed all data.
A part of the results (spontaneous and VUAA1-dependent [Ca2+] increases mediated by
heterologously expressed MsexORCO) was published in: Nolte A, Funk NW, Mukunda L,
Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M (2013) In situ tip-recordings
found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in
Manduca sexta. PLoS One 8 (5):e62648
A similar figure like Fig. 103 appeared in (Nolte et al. 2013).
Unless stated otherwise, all parts of this thesis were implemented by the author.
XVI
List of abbreviations
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007
7TM
4-AP
8-Br-cAMP
8-Br-cGMP
~
[Ca2+]e
[Ca2+]i
Δφ
τ
βγ
a1 and a2
AA
ab
Agam
at
AC
ACh
AChE
AFP
AKAP
AL
AM
AME
AMAE
AMMC
AMPA
AN
ANe
ANF
ANOVA
AOC
AOTU
AP
ATP
AUC
BAL
bHLH
BK
BL
Bmor
8-p-chlorophenylthio-cAMP (EPAC-specific cAMP analog)
seven-transmembrane domains
4-aminopyridine
membrane permeable, hydrolysis-resistant cAMP analog
membrane permeable, hydrolysis-resistant cGMP analog
used in tables to indicate an oscillation
extracellular Ca2+ concentration
intracellular Ca2+ concentration
phase shift of the oscillation
endogenous period length
βγ-subunit of trimeric G protein
arborization area 1 and 2 of PDF-ir neurons of R. maderae
amino acid
antennal basiconic sensillum of D. melanogaster
Anopheles gambiae
antennal trichoid sensillum of D. melanogaster
adenylyl cyclase
acetyl choline
acetyl choline esterase
anterior fiber plexus
A-kinase anchoring protein
antennal lobe
acetoxymethyl ester
accessory medulla (singular)
accessory medullae (plural)
antennal mechanosensory and motor center
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
antennal nerve
anterior neurons (associated with the AME)
atrial natriuretic factor
analysis of variance
anterior optic commissure
anterior optic tubercle
action potential
adenosine trisphosphate
area under the curve
(E,Z)-10,12-hexadecadienal (bombykal)
basic helix-loop-helix
large conductance, calcium-activated K+ channel
basal lamina
Bombyx mori
XVII
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BSA
BSA(-)
bZIP
C-15
CA
CaM
cAMP
CATSPER
CBL/CBU
cGMP
CHO
CICR
clk
ck2
CLNM
CNG
CT
CRE
CREB
cry
CU
CV
cVA
cwo
cyc
CX
DAG
DAPI
dATP8b
dbt
DCV
DD
ddH2O
DEET
DFVNe
DH31
DH31R
disco
DIV
Dmel
DMEM
DmGluRA
DMS
DMSO
bovine serum albumin
essentially fatty acid-free BSA
basic leucine-zipper
(E,Z)-11,13-pentadecadienal (E11,Z13-15:AL)
calyx of the mushroom body
calmodulin
cyclic adenosine monophosphate
cation channels of sperm
lower/upper division of the central body
cyclic guanosine monophosphate
Chinese hamster ovary (cell line)
Ca2+-induced Ca2+ release
clock (gene)
casein kinase 2 (gene)
cell line nutritive medium
cyclic nucleotide gated ion channel
circadian time
cAMP response element
cAMP response element binding protein
cryptochrome (gene)
cuticle
coefficient of variation (standard deviation divided by the mean)
11-cis-vaccenyl acetate
clockwork-orange (gene)
cycle (gene)
central complex
diacylglycerol
4'-6-diamidino-2-phenylindole
P4-type ATPase (a phospholipid flippase) of D. melanogaster
doubletime (gene)
dense core vesicle
constant darkness
bidestilled water
N,N-diethyl-m-toluamide (insect repellent)
distal frontoventral neurons (associated with the AME)
diuretic hormone 31
diuretic hormone 31 receptor
disconnected (gene)
days in vitro
Drosophila melanogaster
Dulbecco’s modified eagle medium
D. melanogaster metabotropic glutamate receptor A
drosomyosuppressin
dimethyl sulfoxide
XVIII
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DN1-3
DNA
dnc
dORKΔ
DT
DUM
E-cells / -peak
EAG
EB
EC50
EDTA
EGTA
EPAC
F340
F380
F340/F380
FaRP
FBS
FSK
Gαs
Gαq
GABA
GDP
GEF
GFP
GL
GluCl
GlutaMAXTM
GOP
GPCR
GPRK2
GR
GRN
GSK-33
GTP
h
H2
HB-eyelet
HBSS
HCN
HEK 293
HEPES
HL
HMA
dorsal neuron groups 1-3
desoxyribonucleic acid
dunce (gene), encodes a cAMP-phosphodiesterase
Drosophila open rectifier K+ channel mutant
distal tract
dorsal unpaired median
evening-cells / -activity peak
electroantennogram
ellipsoid body
half maximal effective concentration
ethylenediaminetetraacetic acid
ethylene glycol tetraacetic acid
exchange protein directly activated by cAMP
fluorescence intensity resulting from excitation at 340 nm
fluorescence intensity resulting from excitation at 380 nm
ratio of the fluorescence intensities F340 and F380
FMRFamide-related peptide
fetal bovine serum
forskolin (stimulates adenylyl cyclase)
α-subunit of a trimeric G protein stimulating adenylyl cyclase
α-subunit of a trimeric G protein stimulating phospholipase C
γ-aminobutyric acid
guanosine diphosphate
guanine nucleotide exchange factor
green fluorescent protein
glial cell
glutamate gated Cl- channel
L-alanine-L-glutamine dipeptide
groom of PDF
G protein-coupled receptor
GPCR kinase 2
gustatory receptor
gustatory receptor neuron
glycogen synthase kinase-3
guanosine trisphosphate
hour
mammalian type 2 histamine receptor
Hofbauer-Bucher-eyelet
Hank's balanced salt solution
hyperpolarization-activated cyclic nucleotide-gated ion channel
human embryonic kidney cell line 293
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hemolymph
5-(N,N-hexamethylene)amiloride
XIX








HR13
HVA
Hvir
IA
ICa
ICa(Cs)
ICa,trans(Cs)
ICa,sust(Cs)





ICl
IFDR
Ih
Ii
Iin,sust


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














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
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
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





IK
IK(Ca)
IKdr
Iout,res
IK,trans
Im
INa
It
Itail
ICU
IgG
IGluR
IK
ILP
IP3
ir
IR
I-V
jet
K(ATP)
K(Ca)
Kir
K(Na)
Kor
KW
l-LNv
LA
LD
LG
H. virescens OR-13 (HvirOR-13)
high voltage-activated
Heliothis virescens
transient (inactivating) A-type K+ outward current
Ca2+ inward current
Ca2+ inward current component in the presence of Cs+
transient (inactivating) Ca2+ inward current component in the presence of Cs+
sustained (non-inactivating) Ca2+ inward current component in the presence
of Cs+
Cl- current
fast delayed rectifier K+ current
hyperpolarization-activated current through HCN channels
ionotropic current
sustained (non-inactivating) inward current counteracting sustained outward
currents
K+ outward current
Ca2+-activated K+ outward current
delayed rectifier-type K+ outward current
residual outward currents in the presence of CsCl and TEA
transient (inactivating) K+ outward current
metabotropic current
Na+ inward current
transduction current
tail current
imaging control unit
immunoglobulin G
ionotropic glutamate receptor
intermediate-conductance Ca2+-activated K+ channel
inferior lateral protocerebrum
inositol 1,4,5-trisphosphate
immunoreactive
ionotropic receptor
current-voltage
jetlag (gene)
ATP-inhibited K+ channel
Ca2+-activated K+ channel
K+ inward rectifier
Na+-activated K+ channel
K+ outward rectifier
Kruskal-Wallis test
large ventrolateral neurons
lamina
light-dark-conditions
lobus glomerulatus
XX
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

































LH
LL
LMS
LNd
LO
LOVT
LPN
LVA
M
M-cells /-peak
MB
MC I-IV
ME
MFVNe
MIA
min
MIP
ML
MNe
mosm/l
MRP
MRT
Msex
MWT
NGS
NMDA
NaChBac
NF1
norpA
NSC
OBP
ODE
OLC
ON
OR
ORC
ORCO
ORN
p1-5
PACAP
PAC(1)
PAS
PB
pb
lateral horn
constant light
leucomyosuppressin
dorsolateral neurons
lobula
lobula valley tract
lateral posterior neurons
low voltage-activated
mol/l
morning-cells / -activity peak
mushroom body
medulla cell group I-IV
medulla
medial frontoventral neurons (associated with the AME)
5-(N-methyl-N-isobutyl)amiloride
minute
myoinhibitory peptide
median lobe
medial neurons (associated with the AME)
milliosmole per liter (number of moles of solute per liter)
multidrug resistance associated proteins
multidrug resistance transporter
Manduca sexta
Mann-Whitney test
normal goat serum
N-methyl-D-aspartate
bacterial Na+ channel
neurofibromatosis 1 gene product
no receptor potential A (gene), encodes a phospholipase C
neurosecretory cell
odorant binding protein
odorant degrading enzyme
ORCO ligand candidate
optic nerve
odorant receptor
orcokinin
olfactory receptor coreceptor (gene: orco)
olfactory receptor neuron
plexus 1-5 of PDF-ir neurons of R. maderae
pituitary adenylyl cyclase-activating polypeptide
specific receptor for PACAP
PER-ARNT-SIM
protocerebral bridge
basiconic sensillum located on the maxillary palps of D. melanogaster
XXI
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

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







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


PBP
PBS
pdp1ε
PDF
PDFAG
PDFCA
PDFLA
PDFME
PDFPI
PDFR
PDH
PDHLI
PED
per
PeTX
PG
PGP
PIP2
PKA
PKC
PKG
PMA
PLC
POC
POT
POTU
pp1
pp2a
PRC
PTU
PTX
PUFA
RAM
rdgA
rdgB








RNA
RNAi
ROI
RT
rut
s -LNv
S2
SAP
pheromone binding protein
phosphate-buffered saline
par domain protein 1ε (gene)
pigment-dispersing factor
PDF-immunoreactive neurons in the abdominal ganglia
PDF-immunoreactive neurons dorsal to the mushroom body's calyx
PDF-immunoreactive lamina neurons
PDF-immunoreactive medulla neurons
PDF-immunoreactive neurons in the pars intercerebralis
PDF receptor
pigment-dispersing hormone
PDH-like immunoreactivity
pedunculus
period (gene)
pertussis toxin
prothoracic gland
P-glycoprotein
phosphatidylinositol 4,5-bisphosphate
protein kinase A
protein kinase C
protein kinase G
phorbol 12-myristate 13-acetate (PKC activator)
phospholipase C
posterior optic commissure
posterior optic tract
posterior optic tubercle
protein phosphatase 1 (gene)
protein phosphatase 2A (gene)
phase response curve
phenylthiourea
picrotoxin (Cl- channel blocker)
polyunsaturated fatty acid
receptor activator molecule
retinal degeneration A (gene), encodes a diacylglycerol kinase
retinal degeneration B (gene), encodes a phosphatidylinositol transfer
protein
ribonucleic acid
RNA interference
region of interest
room temperature
rutabaga (gene), encodes an adenylyl cyclase
small ventrolateral neurons
Drosophila Schneider 2 cells
summed action potential
XXII
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SD
SEM
SF9
SCN
SE
Sfru
sgg
sHBSS
SK
SL
slimb
slo
slob
SLP
SMP
SNMP
sNPF
SPA
SSR
T
TAG
TE
TEA
tim
tim 2
TO
TR
TRP
TTFL
TTX
tyf
UTT
V
Vhold
Vrev
VRMP
VASC
VACC
VIP
VL
VLP
VMNe
VNe
VPAC1/VPAC2
standard deviation
standard error of the mean
Spodoptera frugiperda 9 cells
suprachiasmatic nucleus
suction electrode
Spodoptera frugiperda
shaggy (gene)
supplemented HBSS (Hank's balanced salt solution)
small conductance, calcium-activated K+ channel
sensillum lymph
supernumerary limbs (gene)
slowpoke K+ channel (gene)
slowpoke binding protein (gene)
superior lateral protocerebrum
superior median protocerebrum
sensory neuron membrane protein
short neuropeptide F
sensillum potential amplitude
single sensillum recording
exogenous period length
terminal abdominal ganglion
thecogen cell
tetraethylammonium
timeless (gene)
timeless 2, timeout (gene)
tormogen cell
trichogen cell
transient receptor potential
transcriptional-translational feedback loop
tetrodotoxin
twenty-four (gene)
unpaired t-test
voltage
holding potential
reversal potential
resting membrane potential
voltage-activated Na+ (sodium) channel
voltage-activated Ca2+ channel
vasoactive intestinal peptide
ventral lobe
ventrolateral protocerebrum
ventromedial neurons (associated with the AME)
ventral neurons (associated with the AME)
receptors binding both VIP and PACAP
XXIII

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VPNe
vri
VUAA1

W7



WGA
WT
ZT
ventroposterior neurons (associated with the AME)
vrille (gene)
ORCO agonist (N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol3-yl)thio)acetamide
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride
(calmodulin inhibitor)
wheat germ agglutinin
wildtype
Zeitgeber time
XXIV
Zusammenfassung
Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Periodenlänge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt
synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg
zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus
steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der
spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere
Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone
der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale
Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem
für periphere Schrittmacher dienten.
Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME
(Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene)
untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige
Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass
Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass
in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing
factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor
im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte
waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff
cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by
cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren
und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische
Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle
sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa)
könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und
Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nichtspezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und
Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus
könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen.
Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des
olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die
Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den
ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten
vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo
nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der
unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen
Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie
und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu
zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der
durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die
Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere
Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet
werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.
XXV
XXVI
Summary
Endogenous clocks can be found in almost all organisms, among them circadian clocks, which
generate a period length of approximately 24 hours. These clocks are synchronized (entrained) with
the environment and provide the organism with the ability to anticipate environmental changes.
Next to the central circadian pacemaker (clock), which governs systemic behavioral and physiological
rhythms, peripheral pacemakers can be found in a multitude of tissues, which control temporal
processes in the function of the respective tissue. The aim of this thesis was a physiological
characterization and comparison of central and peripheral insect circadian pacemaker neurons.
Accessory medulla (AME) neurons of the cockroach Rhyparobia maderae were used as model system
for central pacemaker neurons, and olfactory receptor neurons (ORNs) of Manduca sexta as model
system for peripheral pacemaker neurons.
Central pacemaker neurons were analyzed in extracellular recordings from the isolated AME
(network level) and in patch clamp experiments performed with primary AME cell cultures (single cell
level). At the network level, regular neuronal activity and changes of high- and low-activity phases
(oscillations) were shown to be characteristic firing patterns. Glutamate was identified as
neurotransmitter of the AME contributing to the widespread inhibitory synaptic interactions. To a
lesser extent, excitatory synaptic interactions were also demonstrated. The neuropeptide pigmentdispersing factor (PDF), which is released by few AME neurons and functions as important coupling
factor, caused inhibitions and activations, or it increased oscillations. Transient as well as long-lasting
activity changes were detected, which were most probably mediated via the second messenger
cAMP. The exchange protein directly activated by cAMP (EPAC) was suggested to be a possible target
of cAMP. At the single cell level, AME neurons were shown to be depolarized and, thus, the majority
remained silent. Analysis of current-voltage relationships and pharmacological treatment indicated
the expression of different ion channels, such as Ca2+, Cl-, K+, Na+, and non-specific cation channels.
Strong, high voltage-activated Ca2+ currents (ICa) were suggested to play an important role for Ca2+dependent neurotransmitter release, oscillations, and spikes. PDF predominantly inhibited distinct
currents (ICa, IK, and INa) but also activated non-specific cation currents (Ih), resulting in depolarizations
as well as hyperpolarizations. It was hypothesized, that simultaneous depolarizing and
hyperpolarizing effects cause rhythmic membrane potential oscillations. This mechanism might be
employed in PDF-dependent synchronization.
The analysis of peripheral pacemaker neurons focused on the characterization of the M. sexta
olfactory receptor (OR) coreceptor (MsexORCO). Other insects' ORCO orthologues were shown to be
required for membrane localization of tuning ORs. They form heteromeric (OR/ORCO) complexes,
which function as ion channels mediating olfactory transduction in heterologous expression systems.
It was hypothesized that MsexORCO functions as pacemaker channel in pheromone-sensitive ORNs
in vivo, which generates subthreshold membrane potential oscillations, rather than being part of an
ionotropic ion channel complex. MsexORCO was heterologously coexpressed with pheromone
receptor candidates in human embryonic kidney (HEK 293) cells. Immunocytochemistry and Ca2+
imaging experiments revealed a very low expression rate of MsexORCO. Nevertheless, the
experiments indicated that MsexORCO functions as spontaneously active, Ca2+-permeable ion
channel, sensitive to the ORCO agonist VUAA1 and probably also to cyclic nucleotides. Additionally,
the pheromone receptor candidate MsexOR-1 was suggested to be the bombykal receptor. The
characterization of MsexORCO in primary M. sexta ORN cell cultures was not accomplished since
these cells did not respond to ORCO agonists or antagonists.
XXVII
XXVIII
Introduction
1 Introduction
1.1 A short introduction into chronobiology
The movement of the planets in the solar system leads to geophysical rhythms, which have a strong
impact on life on earth. Prominent examples are the 24 hour day/night-cycle caused by the earth’s
rotation around its axis, the alternation of the seasons caused by the inclination of the earth’s axis to
the ecliptic and its revolution around the sun, or the tides caused by the gravitational forces of the
moon and the sun. Adaptations to these environmental changes can be found in most, if not all
eukaryotic organisms in form of endogenous (or biological) clocks, which rhythmically orchestrate
biochemical, physiological and behavioral activities. Endogenous clocks help to anticipate
environmental changes and, therefore, enable the organism to start an appropriate response before
the change actually happens. This provides an advantage in the coordination of the organism's
activities with the periodic environment. The scientific field dealing with this subject is called
chronobiology (ancient Greek: chrónos = time).
One can categorize endogenous rhythms depending on their period length. Circadian rhythms (Latin:
circa = approximately, dies = day) such as the daily sleep/wake cycle have a period length of
approximately 24 hours. Ultradian rhythms (Latin: ultra = beyond) have a period length smaller than
24 hours and can be found for example in the rhythmic generation of spontaneous action potentials
(APs) by pacemaker neurons in the millisecond range, caused by rhythmic depolarizations of the
membrane potential. On the other hand there are infradian rhythms (Latin: infra = below) with
period lengths extending 24 hours, such as the human menstrual cycle with a period length of
approximately 28 days or circannual rhythms (Latin: annus = year) with a period length of
approximately one year, such as bird migration or the adult emergence of the cockchafer Melolontha
melolontha each year around May. Even period lengths bigger than one year are common.
Prominent examples are periodical cicadas with prime periodicities of 13 or 17 years (Grant 2005).
Best studied are circadian clocks, which generate a rhythm matching the 24 hour day/night cycle of
the environment and enable the organism to coordinate its daily change of rest and activity.
Supported by a circadian clock the organism can avoid bad environmental conditions and place its
daily activity phase in a time window providing the most suitable conditions. It is obvious that a
circadian clock provides a benefit in evolution and thus, it is not surprising that circadian clocks were
found in all eukaryotic organisms investigated so far and even in simpler organisms like
cyanobacteria (Reviews: Johnson et al. 1996; Golden et al. 1997). Indeed, a clock with a period similar
to that of the light/dark cycle was shown to enhance the relative fitness under competition in
cyanobacteria (Ouyang et al. 1998) and fruit flies with perturbed circadian clock (see 1.3) were
shown to have around 40 % less progeny than wildtype (WT) flies, indicating a lower reproductive
fitness (Beaver et al. 2002).
First indications for an endogenous circadian clock were provided by the French geophysicist and
astronomer Jean-Jacques d'Ortous de Mairan in the 18th century. He found that the daily leaf
movements of a mimosa continued in constant darkness (de Mairan 1729). Even if de Mairan drew
different conclusions from his experiments, this is the first documented chronobiological experiment
1
Introduction
demonstrating the existence of an endogenous clock. More than 200 years later pioneer work
particularly performed by Jürgen Aschoff and Colin S. Pittendrigh contributed to substantial
chronobiological knowledge (Reviews: Aschoff 1984; Pittendrigh 1993). Finally, the discovery of the
clock genes, initiated by the work of Ronald J. Konopka and Seymour Benzer (1971), and the
application of improved genetic tools especially in the fruitfly Drosophila melanogaster facilitated
major progress in the field (see 1.3).
1.2 General characteristics of circadian clocks
A circadian pacemaker generates a self-sustained oscillation with a period close to, but not exactly
24 hours. This oscillation persists under constant conditions (constant darkness and temperature)
and enables the organism to free-run with its endogenously generated, circadian period (τ).
Depending on the endogenous period, the onset of activity will be timed somewhat earlier (for τ <
24 hours) or somewhat later (for τ > 24 hours) each consecutive cycle (Fig. 1). The endogenous
period is genetically determined and temperature compensated (Pittendrigh and Caldarola 1973). It
is very stable for an adult animal, but it is sensitive to the amount of light in a critical time window
during the animal’s development (Page 1990). Here, it can be set according to the exogenous period
length (T), even to values highly differing from 24 hours. Under free-running conditions the time is
described as circadian time (CT) in circadian hours. Consequently, the size of one circadian hour
depends on τ and usually does not exactly match 60 minutes. The beginning of the activity phase is
defined as CT 0 in diurnal animals or as CT 12 in nocturnal animals. The time window from CT 0 to
CT 12 is called the subjective day, and the one from CT 12 to CT 0 the subjective night (Fig. 1, Fig. 2).
Fig.
1.
Free-running
activity
and
arhythmicity. A. The locomotor activity of a
nocturnal animal is shown (black bars),
which starts running at ZT 12 (= lights off)
and stops running at ZT 0 (= lights on). In
constant darkness (day 6 - 10) the locomotor
activity starts somewhat earlier every day
(= free-running activity), reflecting the
endogenous period length of the pacemaker,
which is smaller than 24 h. Begin of the
activity is defined as CT 12. B. Disruption of
the endogenous pacemaker at day 6 results
in arhythmicity. The figure was redrawn and
modified after (Penzlin 2005).
Under cyclic environmental conditions the clock is synchronized (entrained) with the environment.
The phase of the endogenous oscillation is synchronized to the phase of the exogenous oscillation
and thus, the clock adopts the exogenous period T. Stimuli used for entrainment are called Zeitgeber.
Prominent Zeitgeber are the daily changes of light and temperature, food availability, and social
interactions (Castillo-Ruiz et al. 2012). The time under cycling environmental conditions is termed
2
Introduction
Zeitgeber time (ZT) and, under the consideration of light as Zeitgeber, ZT 0 is defined as the
beginning of the light phase. The influence of a Zeitgeber or internal stimuli on the pacemaker can be
described by phase response curves (PRCs), which show the phase shift of the oscillation (Δφ in
hours) as a function of the phase i.e. the circadian time, at which the stimulus was presented (Fig. 2).
In diurnal as well as nocturnal animals a very similar PRC with a typical biphasic shape is obtained for
light pulses (Page and Barrett 1989). While light has no effect during the subjective day, it results in a
phase delay when given in the early subjective night and a phase advance when given in the late
subjective night (Fig. 2). By this mechanism the endogenous pacemaker is synchronized with the
external light/dark cycle.
Fig. 2. Light-dependent phase-shifts of the locomotor activity. A. Free-running activity of three nocturnal animals (a, b,
and c) is shown for ten days (black bars). The animals are stimulated with light pulses at different CTs. While the light pulse
at CT 6 does not shift the locomotor activity (a), the light pulse at CT 14 results in a phase delay of 5 h (b) and the light
pulse at CT 21 in a phase advance of 4 h (c). B. Plotting of stimulus-dependent phase shifts against the circadian time, at
which the stimuli were delivered, results in a phase response curve (PRC), showing phase advances as positive and phase
delays as negative phase shifts. Stimulation with light pulses results in a typical biphasic PRC. Redrawn and modified after
(Penzlin 2005).
The basic function of a circadian clock can be described by a simple model, first proposed by
Pittendrigh and Bruce (1957). An endogenous pacemaker generates a rhythm and controls effectors
like biochemical, physiological and behavioral activities via output pathways, which may feed back to
the clock. Zeitgeber are perceived by the appropriate receptors, and the information is fed into the
3
Introduction
clock via entrainment pathways to synchronize the endogenous with the exogenous oscillation
(Fig. 3).
Fig. 3. Simplified model of the circadian clock. Rhythmic external stimuli (Zeitgeber) are detected via specific receptors and
used to synchronize the endogenous pacemaker with the environment (entrainment). In turn, the endogenous pacemaker
controls daily physiological and behavioral rhythms such as activity-rest-cycles. The effector actions as well as the
pacemaker might feed back to the entrainment pathways. Redrawn and modified after (Penzlin 2005).
1.3 The central circadian clock of the fruit fly Drosophila
melanogaster
The central circadian clock of the fruit fly Drosophila melanogaster is by far the best studied circadian
system. The high accessibility to genetic manipulations has offered a wide variety of experimental
possibilities, resulting in fast growing knowledge on detailed molecular and cellular mechanisms
underlying rhythmic circadian behavior (Review: Hamilton and Kay 2008; Hardin 2011; Yoshii et al.
2012; Muraro et al. 2013b).
1.3.1 Molecular rhythms are generated by transcriptionaltranslational feedback regulation
Molecular circadian rhythm research began with the discovery of the clock gene period in 1971
(Konopka and Benzer 1971). After treatment with the mutagen ethyl methane sulfonate, Konopka
and Benzer could isolate three mutant fly lines showing severe impairments of circadian behavior
(Fig. 4). One mutant was arhythmic (per0), one showed a shortened period length of 19 h (pers), and
the third showed a longer period length of 28 h (perl). All three mutations could be traced to one
gene located on the X chromosome, which was termed period (per). Remarkably, these were the first
experiments showing that mutations of a single gene can affect behavior (Konopka and Benzer 1971).
Later the per0 and pers mutation could be mapped to single nucleotides of the per gene. A single
nucleotide substitution resulted in a nonsense mutation in the case of per0 and a missense mutation
in the case of pers (Yu et al. 1987). More than 20 years later the next clock gene, timeless (tim), was
discovered (Sehgal et al. 1994). Then, in closer succession dClock (clk, Allada et al. 1998), cycle (cyc,
4
Introduction
Rutila et al. 1998), cryptochrome (dcry, in this thesis termed cry1, Emery et al. 1998; Stanewsky et al.
1998), double-time (dbt, Kloss et al. 1998; Price et al. 1998), vrille (vri, Blau and Young 1999),
timeless 2 (timeout, tim2, Benna et al. 2000), par domain protein 1ε (pdp1ε, McDonald and Rosbash
2001), shaggy (sgg, Martinek et al. 2001), casein kinase 2 (ck2, Lin et al. 2002b), supernumerary limbs
(slimb, Ko et al. 2002), protein phosphatase 2A (pp2A, Sathyanarayanan et al. 2004), jetlag (jet, Koh
et al. 2006; Peschel et al. 2006), protein phosphatase 1 (pp1, Fang et al. 2007), clockwork-orange
(cwo, Kadener et al. 2007; Lim et al. 2007; Matsumoto et al. 2007), and twenty-four (tyf, Lim et al.
2011) were found (Tab. 1).
Fig. 4. Mutations in the period gene strongly affect the period of locomotor activity. A-D. The locomotor activity
(symbolized by black bars) of four individual fruit flies with different genotypes is shown for five consecutive days in
WT
constant darkness. The fly with the wildtype period gene (per ) has a period length of 23.5 h (A), while the fly carrying the
s
l
per mutation has a shortened period length of 19.5 h (B), and the fly with the per mutation a long period length of 28 h
0
(C). The fly with the per mutation does not show rhythmic locomotor activity (D). Redrawn and modified after Konopka
and Benzer (1971).
Molecular rhythms in the clock cells are generated via different interconnected transcriptionaltranslational feedback loops, in which positive elements activate the expression of negative
elements, which feed back and repress the action of the positive elements (Sandrelli et al. 2008,
Fig. 5). In the core feedback loop the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) transcription
factors CLK and CYC form a heterodimer (CLK:CYC) and activate transcription of the genes per and
tim via binding to the genes' E-boxes (Hao et al. 1997; Allada et al. 1998; Darlington et al. 1998; Rutila
et al. 1998). The proteins PER and TIM accumulate in the cytoplasm and form a complex with the
kinase DBT, which translocates into the nucleus (Curtin et al. 1995; Gekakis et al. 1995; Kloss et al.
1998; Kloss et al. 2001). Here, PER mediates DBT-dependent phosphorylation of CLK leading to a
repression of E-box binding and thus to an inhibition of PER's and TIM's "own transcription" (Lee et
al. 1999; Kim and Edery 2006; Yu et al. 2006). Therefore, an oscillation in the mRNA and protein
levels of per and tim is generated (Hardin et al. 1990; Zerr et al. 1990; Sehgal et al. 1995; Zeng et al.
1996).
Like CLK and CYC, PER also contains a PAS domain, which appears to be required for dimerization of
PER and TIM or CLK and CYC (Huang et al. 1993). The functional significance of the PER-TIMdimerization with respect to the nuclear entry is not yet clear. In different studies the heterodimers
were shown to dissociate before nuclear entry (Meyer et al. 2006), PER and TIM were shown to
accumulate in the nucleus with different kinetics (Shafer et al. 2002; Rieger et al. 2006), and PER was
shown to be a more potent inhibitor of CLK:CYC-mediated transcription, if TIM was absent
(Rothenfluh et al. 2000). However, in other studies TIM was shown to be required for nuclear entry
of PER (Saez and Young 1996; Saez et al. 2011), and TIM protected PER from degradation and thus
plays an important role for stabilization of PER (Kloss et al. 2001). For both, PER and TIM a circadian
rhythm in their phosphorylation status was shown (Edery et al. 1994; Zeng et al. 1996), which
5
Introduction
appears to be the major determinant for nuclear entry and stability, with hypophosphorylated forms
being more stable than hyperphosphorylated forms, which precede and most probably cause
degradation (Price et al. 1998; Kloss et al. 2001; Martinek et al. 2001; Stoleru et al. 2007). Thus,
several kinases and phosphatases balance the phosphorylation status of the feedback loop
components (Tab. 1). Apparently these posttranslational modifications allow for a characteristic
delay of up to eight hours between peak levels of per and tim mRNA and protein, which is a
prerequisite for the period length of 24 h (Hunter-Ensor et al. 1996; Hamilton and Kay 2008; Hardin
2011). Additionally, translational control might contribute to this delay. For example, the lateral but
not the dorsal clock neurons express TYF (Lim et al. 2011), which was shown to interact with per and
tim mRNA and translational components such as the 5'-cap-binding complex and poly(A)-binding
protein, suggesting that TYF is a translational activator. Probably translation of PER and TIM is first
inhibited by yet unidentified translation repressors and then promoted by translational activators
such as TYF (Lim et al. 2011).
Next to the core feedback loop other interlocked feedback loops exist (Fig. 5). Not only expression of
PER and TIM, but also expression of the basic leucine zipper (bZIP) transcription factors VRI and
PDP1ε are regulated by the CLK:CYC heterodimer (Blau and Young 1999; McDonald and Rosbash
2001). Vri mRNA and protein levels peak at ZT 14, at about the same time as per and tim mRNA
levels, while peak levels of pdp1ε are delayed (mRNA: ZT 18, protein: ZT 21, Review: Hamilton and
Kay 2008). Both proteins translocate into the nucleus and bind to specific sites in the clk promoter.
While clk transcription is inhibited by VRI binding, it is activated several hours later by PDP1ε binding
(Cyran et al. 2003; Glossop et al. 2003), resulting in oscillations of clk mRNA with peak levels around
ZT 0 (Review: Hamilton and Kay 2008). In contrast to CLK, the gene of its dimerization partner CYCLE
is not rhythmically expressed, although its name may suggest otherwise (Rutila et al. 1998). While
one study could show circadian oscillations in the CLK protein levels employing Western blots (Lee et
al. 1998), another study employing immunohistochemistry found constitutive high CLK levels
throughout the day (Houl et al. 2006), indicating a non-identified posttranscriptional or
posttranslational regulation mechanism leading from rhythmic mRNA levels to constant protein
levels. However, regulation of CLK:CYC mediated transcription certainly depends on CLK's
phosphorylation status with hyperphosphorylated CLK shown to be degraded (Kim and Edery 2006;
Yu et al. 2006).
In addition to VRI and PDP1 the bHLH orange-domain transcription factor CWO was shown to be
CLK:CYC-dependently expressed and to exert negative feedback (Fig. 5). Cwo mRNA reaches its peak
levels around ZT 12 - 15 (Hamilton and Kay 2008) and the protein represses CLK:CYC-mediated
transcription via binding to its target genes' E-boxes including cwo itself. Thus, CWO competes with
CLK for the binding site, apparently leading to high amplitude oscillations of the target genes
(Kadener et al. 2007; Lim et al. 2007; Matsumoto et al. 2007). However, another study found an
inhibitory effect of CWO on its own transcription but activating effects on transcription of per, tim,
vri, and pdp1 (Richier et al. 2008). It was suggested that CWO could activate transcription in the
evening when the nuclear PER level is low, and inhibit transcription in the morning, when the nuclear
PER level is high (Richier et al. 2008).
6
Introduction
Regulators
Interconnected
loops
Core loop
Tab. 1. Clock genes of the fruit fly D. melanogaster (modified after Hamilton and Kay 2008)
Clock gene
cycle (cyc)
dClock (clk)
Expression
constitutive
rhythmic
period (per)
rhythmic
timeless (tim)
rhythmic
Dimerization with PER, stabilization of PER,
target of CRY1-dependent light input
clockwork
orange (cwo)
rhythmic
par domain
protein 1ε
(pdp1ε)
vrille (vri)
rhythmic
casein kinase 2
(ck2)
cryptochrome
(dcry/cry1)
?
double-time
(dbt)
jetlag (jet)
constitutive
protein
phosphatase 1
(pp1)
protein
phosphatase 2A
(pp2A)
shaggy (sgg)
?
bHLH orange-domain transcription factor,
inhibition (activation) of CLK-CYC mediated
transcription via E-box binding
bZIP transcriptions factor, activation of clk
(and possibly cry1) transcription, clkindependent regulation of circadian output
bZIP transcriptions factor, repression of clk
and cry1 transcription
Phosphorylation of PER and TIM, promotion
of nuclear translocation of PER and TIM
Blue-light photoreceptor expressed in
pacemaker cells, light-entrainment,
interaction with PER, TIM, and JET,
mediation of light-dependent depolarization
in a TIM-independent manner, function in
the core feedback loop in peripheral clocks.
Phosphorylation of PER and CLK, promotion
of PER- and CLK-degradation
F-box protein, transmission from lightsignals from CRY1 to TIM, targeting TIM for
degradation
Dephosphorylation of TIM, stabilization of
TIM (and PER)
supernumerary
limbs (slimb)
?
timeless 2
(tim2)
?
twenty-four
(tyf)
?
rhythmic
rhythmic
?
?
constitutive
Characteristics
See clk
bHLH-PAS-transcription factor, dimerization
with CLK, activates transcription of per, tim,
pdp1, and vrille via E-box binding
Dimerization with TIM via PAS-domain,
inhibits CLK-CYC-mediated transcription
Dephosphorylation of PER and CLK,
promotion of PER-stability and nuclear
translocation
Glycogen synthase kinase-3 (GSK-3)
orthologue, regulation PER-TIM nuclear
translocation via phosphorylation of TIM,
interaction with CRY1
F-box/WD40-repeat protein, component of
the ubiquitin-proteasome pathway,
targeting PER for degradation
Mammalian TIM orthologue, requirement
for chromosome integrity, residual function
in light-entrainment
Promotion of PER translation
7
Reference
(Rutila et al. 1998)
(Allada et al. 1998; Bae et al. 1998;
Darlington et al. 1998; Lee et al. 1998,
1999; Houl et al. 2006)
(Konopka and Benzer 1971; Yu et al.
1987; Hardin et al. 1990; Edery et al.
1994; Gekakis et al. 1995; Hao et al.
1997; Meyer et al. 2006)
(Sehgal et al. 1994; Sehgal et al. 1995;
Hunter-Ensor et al. 1996; Myers et al.
1996; Zeng et al. 1996; Naidoo et al.
1999)
(Kadener et al. 2007; Lim et al. 2007;
Matsumoto et al. 2007; Richier et al.
2008)
(McDonald and Rosbash 2001; Cyran
et al. 2003; Benito et al. 2007; Zheng
et al. 2009)
(Blau and Young 1999; Cyran et al.
2003; Glossop et al. 2003)
(Lin et al. 2002b; Akten et al. 2003; Lin
et al. 2005; Meissner et al. 2008)
(Emery et al. 1998; Stanewsky et al.
1998; Ceriani et al. 1999; Emery et al.
2000a; Emery et al. 2000b; Krishnan
et al. 2001; Lin et al. 2001; Rosato et
al. 2001; Cyran et al. 2003; Collins et
al. 2006; Yoshii et al. 2008; Fogle et al.
2011)
(Kloss et al. 1998; Price et al. 1998;
Kim and Edery 2006; Yu et al. 2006)
(Koh et al. 2006; Peschel et al. 2006)
(Fang et al. 2007)
(Sathyanarayanan et al. 2004; Kim and
Edery 2006; Fang et al. 2007)
(Martinek et al. 2001; Stoleru et al.
2007)
(Ko et al. 2002)
(Benna et al. 2000; Benna et al. 2010)
(Lim et al. 2011)
Introduction
Fig. 5. Transcriptional-translational feedback regulation in central clock neurons of D. melanogaster. Central elements of
the interconnected feedback loops are the transcription factors CLOCK (CLK) and CYCLE (CYC), which activate transcription
of the genes period (per), timeless (tim), vrille (vri), par domain protein 1 ε (pdp1), and clockwork orange (cwo). PER and
TIM form a complex with the kinase DOUBLETIME (DBT), which translocates to the nucleus and inhibits CLK/CYC-mediated
transcription. TWENTY-FOUR (TYF) promotes translation of per and tim mRNA. Next to DBT, the kinases SHAGGY (SGG) and
CASEIN KINASE 2 (CK2) and the PROTEIN PHOSPHATASES 1 and 2A (PP1 and PP2A) regulate the phosphorylation status of
the PER/TIM complex. In the second loop VRI inhibits, while PDP1 activates transcription of clk by binding to the gene's
V/P-box. In the third loop CWO inhibits CLK/CYC-mediated transcription. Redrawn and modified after (Tomioka et al.
2012).
1.3.2 Molecular mechanisms underlying light entrainment
Light entrainment of D. melanogaster's central circadian clock mainly functions via the blue-light
photoreceptor CRY1 (Emery et al. 1998; Stanewsky et al. 1998; Emery et al. 2000a; Emery et al.
2000b), but also via classical entrainment pathways (i.e. via complex eyes, ocelli, and HofbauerBucher-eyelet (HB-eyelet). Only if all entrainment pathways are abolished, light entrainment does
not occur (Helfrich-Förster et al. 2001). Light resetting of the clock is mediated via light-dependent
degradation of TIM (Hunter-Ensor et al. 1996; Myers et al. 1996; Zeng et al. 1996). In the CRY1dependent pathway TIM degradation is mediated via an interaction between light-activated CRY1
and TIM (Ceriani et al. 1999; Busza et al. 2004), leading to phosphorylation and subsequent
ubiquitination of TIM (Naidoo et al. 1999; Lin et al. 2001). Apparently these processes are mediated
by the F-box protein JET, which binds phosphorylated TIM and targets it for ubiquitination and
subsequent proteasomal degradation (Koh et al. 2006; Peschel et al. 2006; Knowles et al. 2009;
Peschel et al. 2009, Fig. 6). Once TIM is degraded, its protective function on PER ceases, leading to
DBT-dependent progressive phosphorylation of PER. Hyperphosphorylated PER is then targeted for
proteasomal degradation by the F-box protein SLIMB (Ko et al. 2002). Cry1 is only expressed in a
subpopulation of clock neurons (all s-LNvs and l-LNvs, 3 LNds, 2 DN1a and 6 DN1ps) with a further
subpopulation (the 5th s-LNv, the l-LNvs, and the 3 LNds) showing the strongest oscillations in CRY1
levels (Yoshii et al. 2008). Interestingly, light-dependent TIM degradation does not differ substantially
between CRY1-negative and CRY1-positive neurons (Picot et al. 2007; Yoshii et al. 2008). It was
suggested that TIM-degradation in CRY1-negative neurons is mediated either via intercellular
communication with CRY1-positive neurons or via other unknown mechanisms involving classical
photoreceptor-based light input pathways (Yoshii et al. 2008).
8
Introduction
Fig. 6. Light-dependent TIMELESS degradation.
Light-activated CRYPTOCHROME (CRY1*) binds
TIMELESS (TIM) and promotes its phosphorylation.
Subsequently both proteins are bound by JETLAG
(JET), whereas the affinity is higher for
phosphorylated TIM. Binding of JET mediates
proteasomal degradation of the proteins. Redrawn
and modified after (Hardin 2011).
Light-dependent TIM degradation provides the molecular base for light-dependent effects on
locomotor activity and thus for the typical biphasic shape of a PRC obtained for light pulses (Fig. 2,
Myers et al. 1996). TIM protein levels increase in the early night, peak around ZT 20, and then
decrease to trough levels (Hunter-Ensor et al. 1996; Hamilton and Kay 2008). During the day, when
TIM levels are low, further degradation of TIM should be virtually negligible. In the early night lightdependent TIM degradation counteracts TIM accumulation and thus phase-delays the molecular
clock. In contrast, in the late night, TIM degradation coincides with already falling TIM levels and thus
phase-advances the molecular clock.
1.3.3 Clock neurons of D. melanogaster
Around 150 neurons in the in the fruit fly's brain express the clock genes per, tim, clk, and cyc, and
can be classified into different groups. Three groups of dorsal neurons are located in the superior
brain (DN1, DN2, and DN3). Three groups of lateral neurons are located in the anterior brain: the
large and small ventrolateral neurons (s-LNvs and l-LNvs) and the dorsolateral neurons (LNds). One
group is located in the posterior brain: the lateral posterior neurons (LPNs, Kaneko and Hall 2000;
Houl et al. 2006; Helfrich-Förster et al. 2007b, Fig. 7). The s-LNvs can be further subdivided into four
neurons expressing the neuropeptide PDF and one PDF-negative neuron, which is situated near the
l-LNvs (the 5th s-LNv). The DN1s can be further separated into 14 posterior located neurons (DN1ps)
and two neurons at a more anterior position (DN1as). Thus, to date one can differentiate at least
nine groups of clock neurons, some of which still represent heterogeneous groups according to
different morphology, neurochemical architecture, and function (Tab. 2, Reviews: Helfrich-Förster et
al. 2007b; Yoshii et al. 2012; Muraro et al. 2013b). In addition to the clock neurons other cells in the
brain also express clock genes. The clock gene per for example was shown to be expressed by
photoreceptors and glial cells (Siwicki et al. 1988; Zerr et al. 1990; Ewer et al. 1992; Kaneko and Hall
2000).
9
Introduction
Fig. 7. Central clock neurons with putative projections in the brain of D. melanogaster. Neurons of all groups except the
large ventrolateral neurons (l-LNvs) project to the dorsal brain and neurons of all groups except the second group of dorsal
neurons (DN2) invade the accessory medulla (AME). Only the l-LNvs and the small ventrolateral neurons (s-LNv) express
PDF. For further details see text as well as Tab. 2. DN1-3: dorsal neuron groups 1-3, LNd: dorsolateral neurons, LPN: lateral
posterior neurons. Redrawn and modified after (Helfrich-Förster et al. 2007b).
1.3.3.1 Clock neurons of D. melanogaster innervate the AME and the dorsal
brain
The arborizations of the clock neurons have two main target areas: the dorsal protocerebrum and
the accessory medulla (AME). All clock neuron clusters except for the l-LNvs project to the dorsal
protocerebrum, where they may interact with each other and innervate downstream targets like
neurosecretory centers (Helfrich-Förster et al. 2007a). The AME of D. melanogaster consists of a
central part and a ventral elongation. While the central part contains in- and output synapses, in the
ventral elongation only postsynapses were found, and thus it appears to be mainly an input area. The
central part of the AME is innervated by all s-LNvs, l-LNvs, LNds, and some DN1s and DN3s, but the
only identified neurons innervating the ventral elongation so far are the l-LNvs (Helfrich-Förster et al.
2007a). The arborizations of the l-LNvs are restricted to the AME and the medulla, where they form a
varicose network. These neurons project via the posterior optic tract to the contralateral side, where
they also innervate AME and medulla, and thus probably couple both pacemaker centers. However,
the ventral part of the AME seems to be innervated only ipsilaterally (Park and Griffith 2006; HelfrichFörster et al. 2007a). The second group of neurons with a possible coupling function is the LNd
cluster, which projects via the dorsal fusion commissure to the contralateral AME to form input
synapses. At least some of the LNds also innervate the ipsilateral AME. Thus, the LNds get input from
the ipsilateral as well as the contralateral AME (Helfrich-Förster et al. 2007a).
10
Introduction
Tab. 2. Clock neurons of the fruit fly
Group
# neurons
AME
4
Dorsal
brain
x
s-LNv
5th sLNv
1
x
l-LNv
4-5
LNd
6
x
LPN
3
x
DN1a
2
x
x
DN1p
15
x
partially
DN2
2
x
DN3
40
x
Coupling
Output
Input
Reference
x
PDF, sNPF,
unidentified
neurotransmitter
ACh, GABA,
glutamate, PDF
x
ACh, ITP
PDF
ACh, Dopamine,
GABA, glutamate,
octopamine,
PDF(?)
PDF
(Helfrich-Förster 1995; Hamasaka
et al. 2007; Helfrich-Förster et al.
2007a; Shafer et al. 2008; Johard
et al. 2009; Lear et al. 2009;
Yasuyama and Meinertzhagen
2010; Umezaki et al. 2011; Lelito
and Shafer 2012)
(Kaneko et al. 1997; HelfrichFörster et al. 2007a; Shafer et al.
2008; Johard et al. 2009; Lear et
al. 2009)
(Helfrich-Förster 1995; HelfrichFörster et al. 2007a; Shafer et al.
2008; Lear et al. 2009; McCarthy
et al. 2011; Shang et al. 2011)
x
x
PDF
x
x
partially ACh,
ITP, NPF, sNPF
?
partially
?
glutamate,
IPNamide
partially
glutamate
?
PDF
partially
glutamate
partially PDF
PDF
PDF
(Helfrich-Förster et al. 2007a;
Shafer et al. 2008; Lear et al.
2009)
(Kaneko and Hall 2000; Shafer et
al. 2006)
(Shafer et al. 2006; Hamasaka et
al. 2007; Shafer et al. 2008; Lear
et al. 2009)
(Shafer et al. 2006; Hamasaka et
al. 2007; Shafer et al. 2008; Lear
et al. 2009)
(Shafer et al. 2008; Lear et al.
2009)
(Hamasaka et al. 2007; Shafer et
al. 2008; Lear et al. 2009)
The number of neurons (# neurons) per brain hemisphere, projections into the dorsal brain or the AME, involvement in
coupling of both AMAE, as well as output and input signals of the neurons are shown. Abbreviations: ACh = Acetylcholine,
ITP = ion transport peptide, NPF = neuropeptide F, PDF = pigment-dispersing factor, sNPF = short neuropeptide F. Modified
after (Muraro et al. 2013b).
1.3.4 Control of rhythmic locomotor activity
1.3.4.1 Fruit flies show activity peaks at dusk and dawn
Wildtype D. melanogaster exhibit a bimodal, crepuscular activity pattern under laboratory
conditions. The activity bout around dawn is called morning (M) activity and the activity bout around
dusk is called evening (E) activity (Fig. 8). When entrained flies are transferred to constant darkness
(DD), this activity pattern continues, demonstrating self-sustained oscillations. However, in many
flies the M activity bout is lost in DD and the flies exhibit an unimodal activity pattern corresponding
to the E peak (Wheeler et al. 1993; Helfrich-Förster 2000; Rieger et al. 2006; Taghert and Shafer
2006). When flies are transferred to constant light (LL) conditions, the period lengthens and with
increasing light intensity the flies become arhythmic due to CRY1-dependent TIM degradation (1.3.2,
Aschoff 1979; Konopka et al. 1989). However, certain impairments of the circadian system like the
cryb mutation (Emery et al. 2000a), overexpression of the genes per, morgue (Murad et al. 2007) or
shaggy (Stoleru et al. 2007, Tab. 1), or blocking chemical synaptic transmission in the LNvs (Umezaki
et al. 2011) allow for rhythmicity under LL conditions.
11
Introduction
Fig. 8. Locomotor activity pattern of D. melanogaster. A. Average activity of four light:dark cycles (LD) of a population of
D. melanogaster. The flies show a crepuscular activity pattern with a morning activity peak (M) and an evening activity peak
(E). The increase of activity is anticipated before light on (ZT 0) or off (ZT 12). B. The population activity is shown for three
days in LD (entrained activity) and five days in constant darkness (DD, free-running activity). The circadian rhythm persists in
DD, but the M-peak is lost after some days. The extrapolated Zeitgeber time in constant darkness is shown in italic letters.
Redrawn and modified after (Hyun et al. 2005).
1.3.4.2 Electrical activity plays a crucial role in the circadian system
Next to functional molecular oscillations of clock proteins (see 1.3.1) the electrical activity of the
clock neurons has been shown to be critical for robust rhythmicity. Electrically silencing the LNvs via
misexpression of K+ channels (D. melanogaster open rectifier K+ channel, dORKΔ, or K+ inward
rectifier, Kir2.1) resulted in behavioral arhythmicity in DD (Nitabach et al. 2002; Nitabach et al. 2005;
Park and Griffith 2006; Wu et al. 2008a; Depetris-Chauvin et al. 2011). Electrical hyperexcitation via
misexpression of bacterial Na+ channels (NaChBac) resulted in complex free-running rhythms with
short and long period lengths (Nitabach et al. 2006; Sheeba et al. 2008c; Sheeba et al. 2008b). One
group reported stopping of PER and TIM oscillations in constitutively silenced LNvs and thus
suggested that electrical activity is required for molecular oscillations and that Ca2+ signals may
provide feedback from resting membrane potential (VRMP) oscillations to molecular oscillations
(Nitabach et al. 2002; Nitabach et al. 2005). However, another group demonstrated that stopping of
molecular oscillations only takes place, if the neurons are silenced during development and
adulthood but not if the neurons are adult-specifically silenced, suggesting that arhythmicity may be
a secondary effect (Depetris-Chauvin et al. 2011). Nevertheless this study demonstrated that
electrical silencing impaired circadian remodeling of the s-LNv terminals, although molecular
oscillations were not disrupted. Analysis of the expression profile of silenced and hyperexcited LNvs
demonstrated that electrical activity bidirectionally affected gene expression resulting in an
"evening-like" expression profile for silencing (hyperpolarization) and a "morning-like" expression
profile for hyperexcitation (depolarization, Mizrak et al. 2012). Therefore, electrical activity was
suggested to be a Zeitgeber imposing the time of day. In addition, several ion channels were shown
to play a role in the circadian output: the BK channel slowpoke (slo) and its binding protein (slob,
Jaramillo et al. 2004; Fernandez et al. 2007), the cation channel narrow abdomen (Lear et al. 2005a),
the K+ channel shaw (Hodge and Stanewsky 2008), the voltage-activated Na+ channel (VASC) para
(Wu et al. 2008b), and an inward rectifier K+ channel (Kir, Ruben et al. 2012). To date the function of
electrical activity in the circadian system of the fruit fly is not clarified and "asking whether electrical
activity acts as a Zeitgeber (i.e. a synchronizing cue) or a clock output might be as unhelpful as asking
12
Introduction
what was first, the chicken or the egg" (Muraro et al. 2013b). Furthermore, Ca2+ signaling and
α-amidation of different neuropeptides play a major role in the generation of rhythmicity (Taghert et
al. 2001; Harrisingh et al. 2007).
1.3.4.3 Properties of the ventrolateral neurons
With the use of the GAL4/UAS system (Giniger et al. 1985; Review: Duffy 2002) it was possible to
ablate specific cells by expression of cell death genes or to restrict a functional molecular clock to
distinct subsets of cells. Thus, use of the GAL4/UAS system as well as several mutant lines facilitated
the analysis of the contribution of specific cells to the control of rhythmic locomotor activity. In
several studies the PDF expressing LNvs (see 1.3.5) were shown to play a major role: Restriction of
per expression to the lateral neurons but not dorsal neurons was necessary and sufficient to drive
free-running rhythms (Ewer et al. 1992; Frisch et al. 1994), while ablation or electrical silencing of the
LNvs disrupted free-running rhythms (Renn et al. 1999; Nitabach et al. 2002).
Physiological studies so far concentrated on the LNvs, more precisely on the l-LNvs (due to their size
and better accessibility, Park and Griffith 2006; Cao and Nitabach 2008; Sheeba et al. 2008c; Sheeba
et al. 2008a; Fogle et al. 2011; Choi et al. 2012; Muraro et al. 2013a; Muraro et al. 2013b). The l-LNvs
exhibit circadian changes in their electrical activity with a higher resting membrane potential and AP
frequency during the subjective day (Sheeba et al. 2008a; Muraro et al. 2013a). The firing mode may
switch between bursting and tonic firing, whereupon bursting occurs more frequently during the day
and is driven by slow, large inward currents mediating VRMP oscillations, which are sensitive to the
VASC-blocker tetrodotoxin (TTX), CoCl2, and Ih current impairments (Sheeba et al. 2008c; Sheeba et
al. 2008a; Fogle et al. 2011; Muraro et al. 2013a; Muraro et al. 2013b). Ipsilateral as well as
contralateral pairs of l-LNvs were shown to fire in synchrony, which was suggested to be mediated
by synchronous synaptic inputs, probably mediated by ACh and GABA (McCarthy et al. 2011).
Moreover, these neurons were shown to be light-sensitive: resting membrane potential and AP
frequency are increased in response to light in a CRY1- dependent but TIM-independent manner
(Sheeba et al. 2008a; Fogle et al. 2011). Consistently, the l-LNvs were shown to play an important
role in the phase advance in response to light in the late night (Shang et al. 2008). Less information is
available for the physiology of the s-LNvs, showing also VRMP oscillations and the highest values for
VRMP around lights-on (Cao and Nitabach 2008; Choi et al. 2012). Electrical activity in their terminals
in the dorsal brain is higher in the morning than in the evening and asynchronous between
contralateral s-LNv pairs (Cao et al. 2013).
The s-LNvs and l-LNvs do not only differ in size and projection pattern, but also have different
expression profiles (Kula-Eversole et al. 2010). In contrast to the l-LNvs, the s-LNvs also display
circadian plasticity in their morphology as well as synaptic contacts, which manifests in a circadian
remodeling of their dorsal terminals, showing a more complex, open conformation during the day
and a close conformation during the night, and a varying number and identity of synaptic partners
(Fernandez et al. 2008; Gorostiza et al. 2014). Furthermore, only the s-LNvs display detectable PDF
cycling in their terminals (see 1.3.5, Park et al. 2000). While the s-LNvs are crucial for the control of
locomotor activity in DD and capable of driving other pacemaker neurons, the l-LNvs appear to play
different roles (Grima et al. 2004; Stoleru et al. 2005; Shang et al. 2008; Sheeba et al. 2010). Different
groups reported dampening of the molecular oscillations in l-LNvs when flies were transferred to DD
13
Introduction
(Helfrich-Förster et al. 2001; Yang and Sehgal 2001; Shafer et al. 2002; Peng et al. 2003; Veleri et al.
2003; Lin et al. 2004; Sheeba et al. 2008b) and there is evidence, that the l-LNvs function in another
circuit, which is driven by the DN2s, and do not contribute to the control of rhythmic locomotor
activity (Stoleru et al. 2005). However, studies on "disconnected" mutant flies (disco), which lack
most of the clock neurons, revealed that flies without LNvs were arhythmic but a single LNv with
projections to the superior protocerebrum was sufficient to drive rhythmicity, even if it was an l-LNv
with misexpressed projections (Dushay et al. 1989; Helfrich-Förster 1998). Thus, under certain
circumstances the l-LNvs appear to have the ability to substitute for s-LNvs.
1.3.4.4 Rhythmicity is driven by a flexible network of different clock neurons
Other neurons than the LNvs were also shown to contribute to free-running rhythmicity (HelfrichFörster 1998). The DNs for example contribute to rhythmicity in LD (Veleri et al. 2003; Klarsfeld et al.
2004), functional LNds are sufficient for rhythmicity in LL (Picot et al. 2007), and the LPNs play a
major role in temperature entrainment (Yoshii et al. 2005; Miyasako et al. 2007). In addition to the
clock neurons, glial cells have been shown to play a role in modulating circadian behavior (Suh and
Jackson 2007; Jackson 2011; Ng et al. 2011).
Several conflicting publications implicated distinct clock neurons in the control of M and E activity
(Grima et al. 2004; Stoleru et al. 2004; Stoleru et al. 2005; Rieger et al. 2006; Murad et al. 2007; Picot
et al. 2007; Stoleru et al. 2007). To date the PDF positive s-LNvs and the CRY1-positive DN1ps are
believed to control the M activity (M cells) and the 5th s-LNv, 3-4 LNds, and possibly CRY1-negative
DN1ps are thought to control the E peak (E cells, Review: Yoshii et al. 2012). The M cells can drive
rhythmicity in DD but not LL and their molecular clock is accelerated by light, while the E cells can
drive rhythmicity in LL but not DD and their clockwork is decelerated by light. However, E cells can
also contribute to M activity and M cells to E activity. Apparently, the initial model of M and E cells
was too simplified. The clock neurons constitute a flexible network of interacting oscillators, which
drive rhythmicity. The properties of different cells in this network are affected in different ways by
environmental conditions and the hierarchical order may change (Yoshii et al. 2012; Muraro et al.
2013b).
1.3.5 The function of the neuropeptide PDF
1.3.5.1 PDF is expressed in the ventrolateral clock neurons
The neuropeptide PDF is expressed only by a few cells in the fruit fly's nervous system: around eight
neurons at the anterior edge of each medulla (PDFMEs), up to four neurons dorsal to the calyx of
each mushroom body (PDFCAs), up to six neurons in the pars intercerebralis (PDFPIs), and six
neurons near the ventral midline of the abdominal ganglia (PDFAGs, Helfrich-Förster and Homberg
1993). Most studies concentrated on the PDFMEs, which were shown to be identical with the perexpressing LNvs (see 1.3.3, Helfrich-Förster 1995).
14
Introduction
The pdf gene was isolated and characterized in 1998 (Park and Hall 1998). Surprisingly, no cycling of
pdf RNA could be detected although the peptide exhibited cycling in the terminals of the s-LNvs with
a peak around lights-on (ZT 1) and a trough around lights-off (ZT 13, Park and Hall 1998; Park et al.
2000). Expression of pdf was shown to be regulated by different clock proteins: CLK and CYC appear
to positively regulate transcription in an indirect, E-box-independent manner, while VRI acts
negatively and CLK-independently on a post-transcriptional level (Blau and Young 1999; Park et al.
2000; Allada et al. 2003). Since null mutations of the genes per and tim did not affect RNA levels but
abolished the cycling of the peptide, these proteins were suggested to act on a post-translational
level (Park et al. 2000). Additional evidence for the requirement of clock genes was provided by
overexpression studies: While ectopic expression of pdf in non-clock neurons with projections in the
dorsal protocerebrum rendered the flies arhythmic, overexpression in clock neurons neither
disrupted PDF cycling nor rhythmicity (Helfrich-Förster et al. 2000).
In the LNvs PDF can be detected in large dense core vesicles (DCVs) in PDF-immunoreactive (PDF-ir)
varicosities, indicating a paracrine release mode (Miskiewicz et al. 2004; Yasuyama and
Meinertzhagen 2010). Released PDF is metabolically inactivated by neprilysin-like peptidases, which
were suggested to play an important role in terminating and thus regulating PDF signaling (Isaac et
al. 2007). Next to PDF-ir DCVs, small, clear vesicles that do not contain PDF can be found in output
synapses of the s-LNvs. Thus, at least the s-LNvs also express a classical neurotransmitter next to PDF
(Yasuyama and Meinertzhagen 2010). Consequently, PDF-independent functions of the LNvs like
regulation of cocaine sensitivity (Tsai et al. 2004) or larval light avoidance (Mazzoni et al. 2005) have
been detected. In addition, classical neurotransmitter release contributes to the effects of PDF
autoreceptor activation (see 1.3.5.4, Choi et al. 2012). However, for the control of circadian
rhythmicity PDF release appears to be the main output signal of the LNvs and the classical
neurotransmitter appears to play only a minor role (Renn et al. 1999; Blanchardon et al. 2001; Wu et
al. 2008a; Umezaki et al. 2011).
PDF cycling in the s-LNv terminals was suggested to indicate rhythmic PDF release from these
neurons, acting as a crucial output signal (Park et al. 2000). Indeed, it was shown that PDF
accumulation affects the M activity bout, providing evidence that PDF secretion sets the activity
phase (Wu et al. 2008b). In addition, there are studies derogating the importance of rhythmic PDF
release: Expression of a fusion protein of atrial natriuretic factor (ANF) and green fluorescent protein
(GFP) in the LNvs abolished PDF cycling in the s-LNv terminals while molecular oscillations of the
clock proteins were not affected and the flies were fully rhythmic, suggesting only a minor role for
PDF cycling (Kula et al. 2006). Similarly, phase-independent, constant PDF receptor (PDFR) activation
(see 1.3.5.4) was sufficient to induce strong but abnormal rhythmicity (Choi et al. 2009).
Apparently, PDF accumulation and release is dependent on the electrical activity of the s-LNvs. In the
morning, when PDF release is believed to be highest, the s-LNv's resting membrane potential and
electrical activity in their dorsal terminals are highest, too (Cao and Nitabach 2008; Cao et al. 2013).
Therefore, it is not surprising, that impairment of the electrical activity abolished PDF cycling:
Silenced neurons displayed constitutive low levels of PDF in the terminals but high levels in the
somata (Nitabach et al. 2005; Depetris-Chauvin et al. 2011), while hyperexcited neurons displayed
constitutive high PDF levels in the terminals (Nitabach et al. 2006), each correlated with severe
impairments of circadian behavior (see 1.3.4).
15
Introduction
1.3.5.2 About 60 % of the clock neurons express the PDF receptor
In 2005 three groups isolated and characterized the PDFR (CG13758, also termed HAN or groom of
PDF, GOP), which is a class II peptide G protein-coupled receptor (GPCR) belonging to the secretin
receptor-like B1 subfamily, typically signaling via Gαs and Ca2+ (Hyun et al. 2005; Lear et al. 2005b;
Mertens et al. 2005). No cycling of pdfr RNA or protein could be detected. Instead it was shown to be
expressed at a steady-state level, which is per-dependent (Lear et al. 2005b; Mertens et al. 2005).
While immunocytochemistry and in-situ hybridizations did not provide reliable results, the
expression pattern could be characterized via PDF-dependent cAMP elevations as well as pdfr
promoter driven expression of a reporter gene (Shafer et al. 2008; Lear et al. 2009; Im and Taghert
2010). The PDFR is expressed in about 60 % of all clock neurons (Review: Taghert and Nitabach
2012), such as all s-LNvs, at least two l-LNvs, most LNds, most DN1s, both DN2s, and most DN3s.
Additionally, it is expressed in many non-clock cells of the brain, such as glial cells of the visual
system, cells between the tritocerebrum and the subesophageal ganglion (Im and Taghert 2010),
cells of the antennal mechanosensory and motor center (AMMC, Vecsey et al. 2013), and cells of the
ellipsoid body (EB) of the central complex (Parisky et al. 2008; Pírez et al. 2013). Outside the brain
PDFR expressing cells were also detected, for example in ureter muscles and in the hindgut and
midgut muscles (Talsma et al. 2012), in pheromone producing peripheral clock cells, the oenocytes
(see 1.3.5.5, 1.5.2, Krupp et al. 2013), in the thoracic ganglia (Vecsey et al. 2013), and in the crop,
suggesting PDF release into the hemolymph (Veenstra et al. 2008).
1.3.5.3 The PDF receptor couples to Gαs and probably also to Gαq
Typically for GPCRs of the B1 subfamily the PDFR has been shown to couple to Gαs and to mediate
PDF-dependent increases in the cAMP-level when expressed heterologously in cell cultures (Hyun et
al. 2005; Mertens et al. 2005), transgenically in larval motor neurons (Vecsey et al. 2013), or in situ
(Shafer et al. 2008; Choi et al. 2012; Duvall and Taghert 2012; Talsma et al. 2012; Duvall and Taghert
2013; Pírez et al. 2013). The components of the signaling cascade are packed to signalosomes, whose
components differ between clock cells: In the PDF-positive s-LNvs (M cells, see 1.3.4) Gαs60A,
adenylyl cyclase AC3, and the A-kinase anchoring protein (AKAP) NERVY are required. In the CRY1positive LNds (E cells, see 1.3.4) AC3 only plays a minor role, and mainly AC78C and two AKAPs
(NERVY and AKAP200) are involved (Duvall and Taghert 2012, 2013). Interestingly, the diuretic
hormone 31 receptor (DH31R), which is expressed in the s-LNvs, also couples to Gαs60A in these
neurons, but requires a different AC isoform, indicating highly specific signalosomes (Choi et al. 2012;
Duvall and Taghert 2012). Next to increases in the cAMP level, PDF-dependent Ca2+ concentration
rises were reported for human embryonic kidney (HEK 293) cells heterologously expressing PDFR
(Mertens et al. 2005). In this study, coexpression of the neurofibromatosis-1 gene product (NF1)
enhanced the sensitivity and the strength of PDF responses and thus, was suggested to be involved in
coupling of PDFR to AC. However, there are studies indicating different signaling cascades for PDF: In
one study PDF was suggested to couple to the RAS/MAPK pathway, regulated by NF1 (Williams et al.
2001), and more recently first evidence was provided for PDFR coupling to Gαq and thus
phospholipase C (PLC) signaling (Agrawal et al. 2013). It was suggested that PDFR may couple to both
Gαs and Gαq. In one study, loss of the pdfr (pdfr5304 flies) surprisingly did not mitigate the effects of
16
Introduction
constant PDFR activation in oenocytes of D. melanogaster, which could indicate unspecific responses
or PDF-binding to a second, yet unidentified PDFR (Krupp et al. 2013).
Further components of the PDF-induced signaling pathway have not been reported so far. However,
typical cAMP targets, such as cyclic nucleotide gated (CNG) ion channels, hyperpolarization-activated
cyclic nucleotide-gated (HCN) cation channel, protein kinase A (PKA), as well as the guanine
nucleotide exchange factor (GEF) "exchange protein directly activated by cAMP" (EPAC, Review:
Gloerich and Bos 2010) will likely be involved (Fig. 9).
Fig. 9. PDF signals via Gαs and probably also via Gαq. A. In clock neurons of D. melanogaster the PDF receptor (PDFR)
couples to a trimeric Gs protein. PDF binding leads to separation of the βγ-subunit from the Gαs subunit, which activates
adenylyl cyclase (AC). Hydrolysis of ATP elevates the cAMP level. cAMP might directly activate ion channels as well as
protein kinase A (PKA), which might also activate or inhibit ion channels or translocate to the nucleus and modulate the
transcriptional translational feedback loop (TTFL). Additionally, the "exchange protein directly activated by cAMP" (EPAC)
might be activated, which activates the monomeric G protein RAP and thereby increases the target range of cAMP. B. In
cells of the subesophageal ganglion, the thoracic ganglion, and the antennal mechanosensory and motor complex of
D. melanogaster PDF signaling is required for the regulation of flight. In these cells the PDFR is suggested to couple to the
Gαq signaling pathway, activating phospholipase Cβ (PLCβ), which catalyzes the hydrolysis of phosphatidylinositol 4,5bisphosphate (PIP2) generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The increase of IP3 activates IP3
2+
2+
receptors, leading to Ca efflux from the endoplasmatic reticulum (ER). In combination with DAG the elevated Ca
concentration might activate protein kinase C (PKC), which in turn could activate or inhibit ion channels or modulate the
TTFL. Additionally, DAG may directly activate ion channels.
17
Introduction
Active PKA might phosphorylate target proteins, such as ion channels, in the cytoplasm or translocate
into the nucleus. It might activate cAMP response element-binding protein (CREB), which binds to
cAMP response elements (CREs) within promoters of different genes, thereby regulating these genes'
transcription. CRE domains in insect clock gene promoters, as shown for the mammalian mPer1 gene
(Hida et al. 2000), would provide an effective mechanism for PDF-dependent gene regulation.
1.3.5.4 PDF signaling is required for robust molecular cycling and synchrony
Electrogenic responses to PDF have not been characterized in detail to date. Constant autoreceptor
activation in the s-LNvs was shown to disrupt VRMP oscillations of the s-LNvs and to depolarize the
cells in the presence or absence of TTX, suggested to block VASCs as well as synaptic transmission
(Choi et al. 2012). Recently, acute PDF application on larval motoneurons transgenically expressing
PDFR was shown to depolarize the membrane and increase the excitability of the cells (Vecsey et al.
2013). Moreover, acute PDF application was shown to depolarize DN1p neurons in an adenylyl
cyclase-dependent but PKA-independent manner, probably via activation of CNG channels (Seluzicki
et al. 2014). However, a detailed characterization of the involved ion channels has not been reported
yet.
How does PDF contribute to control circadian rhythms? The behavioral phenotypes for ablation of
the PDF expressing LNvs (Renn et al. 1999; Blanchardon et al. 2001; Shang et al. 2008), electrical
silencing of these neurons (Nitabach et al. 2002; Wu et al. 2008a; Depetris-Chauvin et al. 2011), PDF
knockdown via RNA interference (RNAi) in the LNvs (Shafer and Taghert 2009), as well as pdfr mutant
flies (Hyun et al. 2005; Lear et al. 2005b; Mertens et al. 2005; Lear et al. 2009; Im and Taghert 2010)
resemble the phenotype of pdf01 mutant flies (Renn et al. 1999; Peng et al. 2003; Lin et al. 2004): In
LD conditions the M anticipation is lacking and the E peak is phase-advanced. In DD the M peak is
absent, the flies display a shortened period length of about 23 h, and the majority of flies become
arhythmic after 2 - 3 days. However, in contrast to pdf01 mutant flies, pdfr mutant flies do not lack
the M peak consistently (Im and Taghert 2010). Molecular oscillations in all clock neurons in pdf01
mutant flies were shown to dampen in DD, which correlated with the dampening of behavioral
rhythmicity. This indicates that PDF is required for robust, high-amplitude cycling in DD (Peng et al.
2003; Klarsfeld et al. 2004). Moreover, PER oscillations in the s-LNvs and LNds of pdf01 mutant flies
were shown to continue in DD, but the s-LNvs were shown to desynchronize relative to each other
and the phase of the LNds to advance starting from the third day in DD. This indicates that PDF is
required for coordination of the phase and thus synchronization of different clock neurons (Lin et al.
2004). More detailed analyses revealed that PDF affects clock cell groups differently: It is required for
synchronous cycling in the s-LNvs and DN1as and lengthens the period of these cells. It also
lengthens the period of the 5th s-LNv and the CRY1-positive LNds, but shortens the period of the
CRY1-negative LNds. PDF is crucial for molecular cycling in the CRY1-positive DN1ps, but does not
obviously affect the phase or period of the CRY1-negative DN1ps, the DN2s, and DN3s (Wülbeck et al.
2008; Choi et al. 2009; Lear et al. 2009; Yoshii et al. 2009). PDF signaling from PDF-positive to PDFnegative cells and thus PDFR activation in PDF-negative cells is particularly important to ensure
robust rhythmicity and to set the period length in DD (Choi et al. 2009; Lear et al. 2009). Constitutive
PDF-autoreceptor activation in the LNvs dose-dependently increases M anticipatory activity, shifts
the daily activity to the subjective morning ("increases morningness"), and shortens the period
18
Introduction
(Choi et al. 2012). Therefore, PDF signaling to both PDF-positive and -negative cells appears to be
implicated in setting the period length in DD. Consistently, male flies have higher pdf expression
levels and show an earlier M peak compared to female flies (Park and Hall 1998; Helfrich-Förster
2000). Recent studies suggested that the effects of PDF on the molecular clock are mediated via PKAdependent mechanisms, either promoting stabilization of PER (Li et al. 2014a) or TIM (Seluzicki et al.
2014). The source of PDF release is important too: Release from the s-LNvs is crucial for maintenance
of rhythmicity in DD, while release from s-LNvs and l-LNvs regulates the period length in DD and the
timing of the evening peak in LD (Shafer and Taghert 2009). Interestingly, PDF levels were shown to
decline age-dependently, correlated with attenuations of TIM cycling, period lengthening, and
reduced rhythmic strength. Since PDF overexpression was shown to suppress these age-dependent
changes, it was suggested that age-dependent PDF decline is responsible for rhythm attenuation of
older flies (Umezaki et al. 2012). PDF is not only important for the synchronization of clock cells in the
brain. It is also required for synchronization of peripheral clocks, as shown for prothoracic glands (PG,
Myers et al. 2003) and oenocytes (Krupp et al. 2013).
1.3.5.5 Additional functions of PDF
Next to its function as synchronization signal in the circadian clock, PDF has been shown to be
required for a multitude of additional functions: geotaxis (Toma et al. 2002; Mertens et al. 2005),
rival-induced prolonged mating (Kim et al. 2013), LNv-circuit development (Gorostiza and Ceriani
2013), development of the flight-circuit and modulation of flight (Agrawal et al. 2013), myotropic
effects like ureter contraction (Talsma et al. 2012), as well as photoperiodic responses (Yoshii et al.
2009). Lack of PDF disrupts male courtship behavior (Fujii and Amrein 2010) and females show
increased PDF expression in response to exposure to the male courtship song (Immonen and Ritchie
2012). Additionally, PDF release of both LNv classes plays a major role in the regulation of sleep and
arousal (Parisky et al. 2008; Shang et al. 2008; Sheeba et al. 2008c; Chung et al. 2009; Sheeba et al.
2010; Pírez et al. 2013). While the l-LNvs promote arousal and less sleep at night (Shang et al. 2008;
Sheeba et al. 2008c), the s-LNvs are implicated in sleep onset and probably inhibit arousal (Sheeba et
al. 2008c; Sheeba et al. 2010). PDF signaling to the LNvs themselves and to cells of the ellipsoid body
of the central complex has been shown to be essential in the sleep-regulation circuit (Parisky et al.
2008; Pírez et al. 2013).
1.3.5.6 Parallels between PDF and VIP signaling
Remarkably, there are striking similarities between PDF signaling in insects and vasoactive intestinal
peptide (VIP) signaling in the suprachiasmatic nucleus (SCN), the circadian clock of vertebrates
(Reviews: Vosko et al. 2007; Meelkop et al. 2011; Taghert and Nitabach 2012). VIP is a 28 amino acid
long peptide, which is expressed in the brain, the peripheral nervous system as well as other body
tissues, such as the digestive tract. In the SCN, considered as the "master clock" in vertebrates, VIP is
expressed by around 10 % of the cells, mainly located in the ventrolateral core region (Ibata et al.
1989; Abrahamson and Moore 2001; Vosko et al. 2007; Taghert and Nitabach 2012). VIP and pituitary
adenylyl cyclase-activating polypeptide (PACAP) both belong to the secretin-superfamily and activate
19
Introduction
the same receptors: VPAC1R and VPAC2R (Ishihara et al. 1992; Lutz et al. 1993; Usdin et al. 1994;
Vosko et al. 2007). VPAC2R expression is highest in the SCN, where it is expressed amongst others by
around 30 % of the VIP-positive cells (Usdin et al. 1994; Kallo et al. 2004b). Thus, VIP also signals via
autoreceptors, as shown for PDF (An et al. 2012; Choi et al. 2012; Taghert and Nitabach 2012). In
contrast to PDF and PDFR, VIP and VPAC2R are both expressed in a circadian manner (Shinohara et al.
1999; Dardente et al. 2004; Kallo et al. 2004a), and similar to PDF, VIP is released in a circadian
manner (Shinohara et al. 2000a; Vosko et al. 2007). While VIP does not show any sequence identity
with the shorter PDF, the PDFR as well as VPAC1R and VPAC2R are related members of the secretin
receptor-like B1 subfamily of GPCRs (Mertens et al. 2005; Vosko et al. 2007; Couvineau and Laburthe
2012). Consistently, the PDFR was shown to be also activated by PACAP-38, when heterologously
expressed in HEK 293 cells (Mertens et al. 2005). As shown for PDFR activation, VPAC2R couples to
Gαs and activation leads to increased cAMP levels and activation of PKA (Rea 1990; Vanecek and
Watanabe 1998; Itri and Colwell 2003; Meyer-Spasche and Piggins 2004; Kudo et al. 2013).
Additionally, VIP signaling was shown to be dependent on AC signaling as well as PLC signaling and it
was suggested that VPAC2R couples to Gαs and Gαq (An et al. 2011), as suggested previously for the
PDFR (Agrawal et al. 2013). Most interestingly, the phenotypes of mice mutants lacking either VIP
(Colwell et al. 2003) or its VPAC2 receptor (Harmar et al. 2002) are very similar to those of pdf or pdfr
null mutant flies (Renn et al. 1999; Peng et al. 2003; Lin et al. 2004; Hyun et al. 2005; Lear et al.
2005b; Mertens et al. 2005; Lear et al. 2009; Im and Taghert 2010): Most of these mice behave
arhythmic in DD and those mice that retain weak rhythmicity, display a shortened period length.
Molecular oscillations of several clock genes display low amplitudes, single cells loose rhythmicity,
and synchrony between different SCN cells is disrupted (Harmar et al. 2002; Maywood et al. 2006).
Additionally, synchronous firing and characteristic rhythms in the neural activity of SCN neurons are
abolished (Cutler et al. 2003; Aton et al. 2005; Brown et al. 2007). Thus, similar to PDF signaling, VIP
signaling is required for synchronous output of the SCN. Beyond the circadian master clock there are
similarities, too: Both peptides have endocrine, myotropic functions in the digestive tract or
urogenital system (Talsma et al. 2012 and references mentioned therein).
1.4 The central circadian clock of the cockroach Rhyparobia
maderae
1.4.1 The compound eyes are required for entrainment
Despite their limited genetic accessibility cockroaches have always been popular model organisms,
mainly due to their size, their long life spans and their high capability of resistance. Therefore, it is
not surprising that many basic studies investigating circadian clock mechanisms were performed on
cockroaches. Indeed, the cockroach R. maderae was the first animal, in which the central circadian
pacemaker was located (Reviews: Page 1990; Homberg et al. 2003).
When cockroaches are kept in running wheels in cycles of 12 hours light and 12 hours darkness (LD
12:12), the animals’ locomotor activity lies predominantly in the night and starts with the transition
from light to darkness. The entrainment of the locomotor activity to the external LD cycle was shown
20
Introduction
to be exclusively dependent on the compound eyes of the animal (Fig. 10). When the compound eyes
were painted with black lacquer or the optical nerves, connecting the optic lobes with the ommatidia
of the compound eyes, were severed bilaterally, animals were shown to free-run in LD conditions. In
contrast, when the ocelli were surgically removed, the locomotor activity rhythm was not affected
(Roberts 1965; Nishiitsutsuji-Uwo and Pittendrigh 1968a; Page et al. 1977; Review: Page 1990).
Therefore the compound eyes but not the ocelli are necessary and sufficient for light entrainment.
Currently it is not known whether light-sensitive molecules like CRY1, expressed in the clock cells
itself and shown to be the predominant light input pathway for the clockwork of D. melanogaster,
are involved in entrainment. For another cockroach species (P. americana) the peak sensitivity for
entrainment was shown to be around 495 nm, thus activating green light photoreceptors but no blue
light photoreceptors, arguing against an involvement of CRY1 (Mote and Black 1981). Furthermore,
for cockroaches, which were free-running due to their painted heads, it was shown that insertion of a
glass window over the protocerebrum was sufficient for entrainment, only if the optic nerves were
intact, suggesting that light influx via the glass window activates molecules in cells distal to the optic
nerves, apparently ommatidial molecules (Nishiitsutsuji-Uwo and Pittendrigh 1968a).
Fig. 10. The compound eyes but not the ocelli are
necessary and sufficient for entrainment in R. maderae. A
single Madeira cockroach was kept in a running wheel for
80 days under LD conditions (12:12 h) and locomotor
activity was recorded. On day 20 both complex eyes were
painted with black lacquer, resulting in a loss of
entrainment and appearing of free-running activity. On day
50 the lacquer was removed, facilitating entrainment to the
LD cycle. On day 68 the ocelli of the cockroach were
surgically removed, which apparently did not affect the
entrained locomotor activity. Redrawn and modified after
(Roberts 1965).
1.4.2 Localization of the pacemaker in the optic lobes
The localization of the central pacemaker controlling locomotor activity was investigated in a series
of lesion and transplantation experiments (Review: Page 1990). Nishiitsutsuji-Uwo and Pittendrigh
(1968b) first suggested that the central pacemaker resides in the optic lobes of the Madeira
cockroach. In their experiments different parts of the nervous system were removed or connections
between different areas were sectioned. The abdominal ganglia, thoracic ganglia, subesophageal
ganglion, and the pars intercerebralis could be excluded as autonomous pacemakers controlling
locomotor activity. For the pars intercerebralis a role in mediating the endogenous rhythm via
21
Introduction
secretion was suggested, but only if connections between pars intercerebralis and optic lobe, as well
as connections between optic lobe and compound eye were intact. If the connections between
midbrain and proximal end of the lobula (= optic stalks) were sectioned bilaterally or both optic lobes
were removed, the rhythmicity in LD conditions was abolished. If only one optic stalk was sectioned
(unilateral section), rhythmicity was not abolished. These results suggested that bilateral
pacemakers, located in the optic lobes, generate a rhythm, which is imposed via neuronal
connections to the pars intercerebralis. The pars intercerebralis was suggested to mediate the
rhythm via hormone release to the thoracic ganglia, which in turn control locomotor activity
(Nishiitsutsuji-Uwo and Pittendrigh 1968b; Review: Page 1990). The effect of bilateral optic stalk
section on the free-running rhythm (i.e. arhythmicity) persisted when the optic lobes were removed,
but the free-running rhythm regenerated in 3 - 5 weeks when the isolated optic lobes remained in
situ. The behavioral regeneration correlated with regeneration of neuronal connections between
optic lobe and midbrain was blocked by the insertion of a glass barrier between optic lobe and
midbrain. It was concluded, that neurons in the optic lobes generate the free-running rhythm and
that the information is transmitted to midbrain targets via the optic tracts (Page 1983b; Review: Page
1990). In these experiments the period of the free-running rhythm (τ) after regeneration was
strongly correlated with the preoperative period. Moreover, when optic lobes from cockroaches
raised in LD 11:11 (T 22) or LD 13:13 (T 26) cycles were removed and transplanted, the free-running
rhythm in some animals was restored in 4 - 8 weeks, correlated with a re-innervation of midbrain
targets, and the postoperative free-running period of the host animal was highly correlated with the
preoperative period of the donor animal (Page 1982). In the optic stalk section experiments it was
further demonstrated that the optic lobe pacemaker controls the phase of the free-running rhythm
(Page 1983b; Review: Page 1990). When the optic lobes remained in situ, still connected to the
compound eyes but isolated from the midbrain, the phase of the subsequent rhythm was conserved
during the regeneration process and it was possible to shift the phase of the subsequent rhythm via
postoperative light exposure. Phase control of the optic lobe was also demonstrated by the effects of
localized low-temperature pulses (Page 1981; Review: Page 1990). When one optic stalk was
sectioned, cooling of the intact optic lobe, but not the isolated one, for 6 h to 7.5 °C starting at
activity onset resulted in a phase delay of several hours. These experiments suggested that the optic
lobes contain pacemakers that control the period as well as the phase of the free-running rhythm.
Such an endogenous oscillator should have the ability to generate an oscillation in a biochemical or
physiological function, even if isolated from the animal. Indeed, a circadian rhythm in spontaneous
activity could be recorded from the descending cervical connectives, the connection between the
subesophageal ganglion and the first thoracic ganglion, which was abolished when both optic lobes
were removed (Colwell and Page 1990; Review: Page 1990). Moreover, it was possible to record a
circadian rhythm in spontaneous activity from the optic stalk of the isolated optic lobe in vitro,
providing direct evidence that the optic lobe accommodates a self sustained oscillator, which
generates a circadian rhythm in the absence of hormonal or neuronal input from tissue outside the
optic lobe (Colwell and Page 1990; Review: Page 1990).
22
Introduction
1.4.3 The bilateral pacemakers are mutually coupled
Experiments, in which either the left or the right optic lobe was completely excised or isolated from
the midbrain, revealed no differences between the left and right optic lobe and thus, demonstrated
that the bilateral optic lobes are functionally redundant (Page et al. 1977; Review: Page 1990). Each
compound eye was shown to have the ability to entrain both oscillators. When the optic nerves were
unilaterally sectioned and the cockroach was subsequently exposed to a T22 or T26 light cycle, both
pacemakers were entrained to the new exogenous period, indicated by the absence of any rhythm
component with the endogenous period (Page et al. 1977; Page 1983a; Review: Page 1990).
Furthermore, unilateral optic lobe lesions resulted in a lengthening of the mean free-running period
from 23.7 h to 23.9 h. These results suggested that both pacemakers are mutually coupled and that
the coupling interactions shorten the period (Page et al. 1977; Review: Page 1990). Additionally, the
localized low-temperature pulse experiments, mentioned above, found evidence for coupling
between the pacemakers. One optic lobe was cooled down and subsequently removed. If the lobe
was removed after four days, the subsequent free-running rhythm was phase shifted. If the lobe was
removed after 30 min, this was not the case (Page 1981). Therefore, cooling did not affect the
contralateral optic lobe directly, but the phase shift information was transmitted subsequently,
suggesting a coupling pathway between the pacemakers (Review: Page 1990).
When optic lobes were unilaterally transplanted between cockroaches raised in T22 or T26 light
cycles, which still had one intact optic lobe, the free-running period of the host animal did not change
(Page 1983a; Review: Page 1990). Not till the intact optic stalk was severed, the donor pacemaker
was shown to control activity, suggesting that the neuronal connections of the donor optic lobe to
the midbrain had regenerated but the intact host pacemaker suppressed the expression of the donor
pacemaker. If the host optic lobe regenerated its neuronal connections, the activity rhythm was
clearly composed of two free-running components. These results showed that under the
experimental conditions severed or transplanted optic lobe pacemakers could regenerate
connections to the midbrain for locomotor activity control but not the coupling pathway between
both pacemakers, suggesting that these two pathways are functionally distinct (Page 1983a; Review:
Page 1990).
1.4.4 Cellular identity of the central pacemaker
1.4.4.1 The accessory medulla and associated PDF-ir neurons
After the first indications that the optic lobes accommodate the pacemakers (Nishiitsutsuji-Uwo and
Pittendrigh 1968b) several experiments were performed to narrow down the area of the pacemaker
(Review: Page 1990). Roberts (1974) removed different parts of the optic lobes or used
microelectrodes to electro-coagulate optic lope tissue at different sites and examined the effects on
the locomotor activity rhythm in LD conditions. In these experiments the pacemaker could be
localized to the medulla-lobula complex and the lamina was excluded. These results were confirmed
with further microlesion experiments performed by Sokolove (1975), who localized the pacemaker to
a somata region near the second optic chiasm between lobula and medulla, and by Page (1978), who
23
Introduction
found the ventral two-thirds of the lobula to be critical for the activity rhythm. However, the cellular
identity of the pacemaker remained elusive until the first immunostainings with an antibody directed
against the crustacean octadecapeptid β-pigment-dispersing hormone (β-PDH) were performed
(Dircksen et al. 1987; Rao and Riehm 1989; Homberg et al. 1991; Nässel et al. 1991). PDH was found
to elicit pigment dispersion in epithelial chromatophores and specific ommatidial pigment cells in
crustaceans, thus playing a role in light adaptation. A related peptide was identified in insects and
was termed pigment-dispersing factor (PDF, Review: Rao and Riehm 1989). Homberg and coworkers
(1991) found three cell groups in the optic lobes of different orthopteroid insects expressing PDH-like
immunoreactivity (PDHLI, for reasons of simplicity insect cells expressing PDHLI will be termed PDF-ir
in the following). Two cell groups lying in the posterior ventral and posterior dorsal edge of the
lamina were termed PDF-ir lamina cells (PDFLAs) and one cell group lying in the anterior edge of the
medulla was termed PDF-ir medulla cells (PDFMEs). It was not possible to clearly separate the
branching pattern, but the PDFLAs appeared to have arborizations in the lamina and the distal
medulla, while the PDFMEs appeared to arborize in the lamina and different midbrain areas.
Additionally, PDFMEs and PDFLAs both appeared to densely innervate a small neuropil at the
ventromedial edge of the medulla that was called accessory medulla (AME, plural: accessory
medullae, AMAE) according to a similar structure in holometabolous insects, which was described as
termination site of larval and extraretinal photoreceptors (Ehnbom 1948; Hagberg 1986; Hofbauer
and Buchner 1989; Homberg et al. 1991; Fleissner and Frisch 1993). It was suggested that the AME
should not necessarily be considered as remnant of the larval visual system but instead as a common
visual insect neuropil that does not show the typical retinotopic organization (Homberg et al. 1991).
The lack of retinotopic organization suggested that the AME is not involved in visual image
processing but instead serves a different purpose (Homberg et al. 1991; Petri et al. 1995; Reischig
and Stengl 1996), which was also supported by intracellular recordings of neurons innervating the
AME (Loesel and Homberg 2001). The PDFMEs fulfilled several anatomical criteria proposed for
circadian pacemaker neurons: Their somata near the AME lay exactly in the area of the optic lobe
that was proposed to accommodate the pacemaker (Roberts 1974; Sokolove 1975; Page 1978;
Homberg et al. 1991). The arborizations in the lamina could allow for visual input and circadian
control of visual sensitivity and the arborizations in the midbrain, possibly contact sites with
descending neurons and neurosecretory cells, are a prerequisite for the control of locomotor activity
and various other physiological and behavioral functions. Moreover, in the cockroach P. americana
prominent PDF-ir commissures were found to innervate the contralateral brain hemisphere while
contralateral projections were almost absent in the crickets Acheta domesticus, Gryllus bimaculatus,
Teleogryllus oceanius and T. commodus (Homberg et al. 1991). This finding correlated well with weak
coupling interactions between the bilateral pacemakers in crickets and the strong coupling in
cockroaches (Loher 1972; Roth and Sokolove 1975; Page et al. 1977; Page 1978; Wiedenmann 1980;
Page 1981; Page 1983a; Wiedenmann 1983, 1984; Stengl 1995; Ushirogawa et al. 1997), suggesting
that PDF-ir neurons also play a role in coupling (Homberg et al. 1991). Therefore, the PDFMEs and
the AME, which is densely innervated by PDF-ir neurons, appeared to be excellent candidates for
circadian pacemaker neurons and the circadian pacemaker center neuropil (Homberg et al. 1991).
24
Introduction
1.4.4.2 Transplantation studies located the pacemaker to the AME
Stengl and Homberg (1994) provided first evidence that PDF-ir neurons are involved in circadian
control of locomotor activity in R. maderae. As described above, severing both optic lobes results in
abolished locomotor rhythmicity but regeneration within several weeks may occur (NishiitsutsujiUwo and Pittendrigh 1968b; Page 1982; Page 1983b). Thus, it was investigated if the regeneration of
rhythmicity correlated with regeneration of PDF-immunoreactivity. Indeed, it was shown that in all
cockroaches, which regained rhythmicity, PDF-ir neurons had regenerated arborizations in their
original target sites in the protocerebrum. Moreover, a correlation between the free-running period
and the number of PDF-ir commissures between both brain hemispheres was found (Stengl and
Homberg 1994). Apparently, a higher number of PDF-ir commissural fibers resulted in a shorter
period, again suggesting a coupling function of PDF-ir neurons and providing for the first time a
cellular basis for the general finding that mutual coupling accelerates the pacemakers (Page et al.
1977; Page 1978). In contrast to earlier studies (Page 1983b, a), this study demonstrated that not
only the output pathways controlling locomotor activity, but also the coupling pathway has the
ability to regenerate. Further evidence for circadian pacemaker function of AME and PDFMEs was
provided by transplantation studies (Reischig and Stengl 2003a). Ectopic transplantation of AMAE
with associated neurons into the antennal lobe of cockroaches, whose optic lobes had been
removed, restored circadian locomotor activity, demonstrating that the circadian pacemaker center
is included in the AME grafts. Correlated with restoration of activity, PDF-ir fibers originating from
large and medium-sized PDFME neurons (see 1.4.7.1) of the transplants innervated original targets in
the midbrain, thus demonstrating that at least some PDF-ir cells are circadian pacemaker neurons.
Rhythmic cockroaches showed regeneration of PDF-ir fibers in the superior median protocerebrum
(SMP) and superior lateral protocerebrum (SLP), presumed output regions to locomotor activity
centers in the cockroach as well as D. melanogaster (Homberg et al. 1991; Renn et al. 1999). Other
target areas like the inferior lateral (ILP) and the ventrolateral protocerebrum (VLP) or the posterior
optic tubercle (POTU) where not necessarily innervated and thus, were suggested to be no
prerequisite for restoration of locomotor activity. The fact that the period was not correlated with
the number of PDFMEs surviving in the AME transplants, indicated that the PDFMEs are not the sole
pacemaker neurons. Indeed, these experiments did not rule out the possibility that other cells
neighboring the PDFMEs are also circadian pacemaker cells, because only PDF-ir cells were
investigated in this study (Reischig and Stengl 2003a).
1.4.4.3 The AME is composed of glomeruli
Meanwhile the AME has more comprehensively been investigated. Much is known about the
morphology of the neuropil and its associated neurons (see 1.4.5), their chemoarchitecture
(see 1.4.7), input-, output-, and coupling pathways (see 1.4.6, 1.4.9), as well as their physiology
(see 1.4.10). The AME is a small pear-shaped neuropil at the frontal medioventral edge of the
medulla. It is located around 80 µm below the anterior surface of the optic lobe and measures
78 - 100 µm along its longitudinal axis in dorso-ventral direction, 34 - 40 µm in lateral, and 31 - 51 µm
in anterior-posterior direction (Petri et al. 1995; Reischig and Stengl 1996). The volume of the AME
was shown to be correlated with volumes of medulla, protocerebral bridge and the upper division of
the central body, suggesting functional connections between these neuropils (Wei et al. 2010). While
25
Introduction
other optic lobe neuropils are retinotopically organized, the AME is composed of glomeruli. Initially,
these structures were differentiated from other glomerular structures, such as the glomeruli of the
antennal lobe (Tolbert and Hildebrand 1981; Homberg et al. 1989), and described as noduli, which
are densities, resulting from the accumulation of intracellular structures (Reischig and Stengl 1996).
One can differentiate between several ventral glomeruli and one bigger frontal glomerulus, which
form the first subdivision, and one large dorsal glomerulus forming the second subdivision (Fig. 11).
The dense glomeruli are embedded in loose interglomerular neuropil, which extends into the distal
tract, and are surrounded by loose shell neuropil. Distally and ventrocaudally the AME is enveloped
by the medulla, frontodorsally the boundary is formed by the anterior fiber network, and dorsally by
the root of the posterior optic tract. The medial, frontoventral, and ventral borders are formed by
the same glial sheath that surrounds the medulla (Reischig and Stengl 1996). Four types of DCVs
were found in different parts of the AME: Granular DCVs were restricted to glomerular neuropil,
apparently formed by local neurons, and small, medium-sized and large DCVs were restricted to
interglomerular and shell neuropil (Reischig and Stengl 1996).
Fig. 11. The accessory medulla is a glomerular
neuropil with high electron-density. In electron
micrographs the accessory medulla (AME)
clearly can be distinguished from surrounding
tissues due to the dense innervation by fibers
containing accumulations of vesicles. The inlay
illustrates the orientation and the glomerular
composition. Abbreviations: LA: lamina, ME:
medulla, LO: Lobula, DGL: dorsal glomerulus,
FGL: frontal glomerulus, VGL: ventral
glomerulus, c: caudal, d: dorsal, f: frontal, l:
lateral, m: medial, v: ventral. Scale bar: 50 µm.
Redrawn and modified after (Reischig and Stengl
1996).
1.4.5 Neurons associated with the accessory medulla
Around 300 neurons appear to have ramifications in the AME and to communicate with each other in
this neuropil (Reischig 2003). According to the position of their somata and morphological criteria
these AME-associated neurons can be classified into several cell groups (Fig. 12): distal frontoventral
neurons (DFVNes, n = 29 ± 10), medial frontoventral neurons (MFVNes, n = 49 ± 7), medial neurons
(MNes, n = 56 ± 12), ventral neurons (VNes, n = 24 ± 5), ventromedial neurons (VMNes, n = 35 ± 5),
ventroposterior neurons (VPNes, n = 36 ± 9), and anterior neurons (ANes, n = ?, Reischig and Stengl
1996, 2003b; Soehler et al. 2008). Additionally, some neurons with somata at a more posterior
position (for example posterior PDFMEs and neurons of the MC III-group) and at least some of the
PDFLAs project into the AME (Homberg et al. 1991; Stengl and Homberg 1994; Petri et al. 1995;
Reischig and Stengl 2002, 2003b).
26
Introduction
1.4.6 Medulla cell groups involved in bilateral coupling
The coupling pathway between the bilateral symmetric AMAE was investigated by the use of
neuronal tracers (Reischig and Stengl 2002; Reischig et al. 2004; Hofer and Homberg 2006a; Soehler
et al. 2011). In these experiments the neuronal tracer was applied either via backfills from the
ipsilateral optic stalk or by injection into the ipsilateral AME. Retrograde and anterograde transport
of the tracer allowed labeling of possibly the complete arborizations of the neurons, which had
absorbed the tracer, and revealed seven commissures (tracts 1-7) connecting both optic lobes. Three
of them (tracts 3, 4, and 7) were well suited to couple both AMAE. All three tracts converge in the
contralateral lobula valley tract (LOVT), which runs from the proximal end of the medulla to the
proximal end of the lobula, where it bifurcates into the anterior optic commissure (AOC) and the
posterior optic tract (POT), which leads to the posterior optic commissure (POC). Tracts 3 and 4 cross
the midbrain via the POC and tract 7 via the AOC. Associated with these tracts two fiber systems
were labeled, which emerge from the contralateral LOVT: the fan-shaped anterior-layer fiber system,
which sends varicose fibers through the anterior layer of the medulla, and the middle-layer fiber
system, which arborizes in middle layers of the medulla. Both fiber systems appeared to innervate
the interglomerular neuropil of the contralateral AME (Reischig and Stengl 2002). Stained somata in
the contralateral medulla, associated with these commissures, were assigned to four cell groups,
termed medulla cell groups I - IV (MC I - IV, Reischig and Stengl 2002; Soehler et al. 2011). The MC I
group consists of five somata, belonging to the VNe group of AME-associated neurons, and innervate
the fan-shaped anterior-layer fiber system (Reischig and Stengl 2002). Two of these VNes additionally
innervate the middle-layer fiber system (Wei et al. 2010). The MC II group consists of up to 35
somata and is identical to the VMNe group. These cells are associated with tract 7, projecting via the
POC, and form a middle-layer fiber system of the medulla. The MC III group counts up to three
somata located at a posterior position similar to the pPDFMEs (Reischig and Stengl 2002) and the MC
IV group consists of up to five neurons, which form a subpopulation of the MNe group (Soehler et al.
2011). The projection pattern of both groups has not been characterized in detail and, in contrast to
MC I and II, it is not clear if neurons of these groups indeed connect both AMAE (Soehler et al. 2011).
1.4.7 Neurotransmitters of the accessory medulla
The neurons innervating the AME show immunoreactivity to several antisera against classical
neurotransmitters and neuropeptides. The classical neurotransmitters include γ-aminobutyric acid
(GABA), acetylcholine (ACh), histamine, and serotonin (Petri et al. 1995; Loesel and Homberg 1999;
Petri et al. 2002; Schendzielorz 2013). The neuropeptides include allatostatin, allatotropin, baratin,
corazonin, different FMRFamide related peptides, gastrin/cholecystokinin, leucokinin I, myoinhibitory
peptide (MIP), orcokinin, and PDF (Petri et al. 1995; Nässel et al. 2000; Petri et al. 2002; Reischig and
Stengl 2003b; Hofer and Homberg 2006b, a; Soehler et al. 2007; Soehler et al. 2008; Schulze et al.
2012). There is not only immunocytochemical evidence for the presence of these substances in the
AME: several of them were detected by mass spectrometric analysis of the tissue. Furthermore,
some of these substances were injected into the head capsule or directly into the vicinity of the AME
of free-running animals to test for possible effects on circadian locomotor activity. Thus, peptidergic
signaling appears to play a key role in the AME, as shown for the mammalian circadian pacemaker
27
Introduction
center (Harmar 2003; Review: Homberg et al. 2003). Remarkably, some of the AME-neurons
coexpress several neuropeptides and apparently employ peptide sorting.
1.4.7.1 Pigment-dispersing factor in the AME
Best characterized among the AME-associated neurons are the PDF-ir neurons with somata in the
lamina (PDFLAs) and the medulla (PDFMEs). PDFLAs can be found at a ventral (vPDFLAs) and a dorsal
position of the lamina (dPDFLAs). Both groups contain 50-70 neurons (Homberg et al. 1991; Stengl
and Homberg 1994; Petri et al. 1995; Wei et al. 2010). Around twelve PDF-ir somata are located
anterior to the AME, which were named anterior PDF-ir medulla neurons (aPDFMEs, Fig. 12, Petri et
al. 1995). According to their soma-size, the intensity of the PDF-immunoreactivity, the DCV content,
and the coexpression of other neuropeptides these neurons can be classified into faintly labeled
small, stronger labeled medium-sized, and intensely labeled large aPDFMEs (Reischig and Stengl
2003b; Soehler et al. 2011). Each subgroup contains about four neurons. While the large and
medium-sized aPDFMEs belong to the VNe group of AME-neurons, which were shown to form output
pathways to different targets in the protocerebrum and coupling pathways to the contralateral optic
lobe, the small aPDFMEs belong to the DFVNe group, which are local neurons of the AME. Because of
their weak PDHLI the small aPDFMEs have not been investigated in detail to date (Reischig and Stengl
2003b). Additionally, about two large and two small PDF-ir somata can be found at a more posterior
position, which were termed posterior PDFMEs (pPDFMEs, Fig. 12, Petri et al. 1995). Since large and
medium-sized aPDFMEs could not be clearly distinguished from each other, the discrimination was
based on additional FMRFamide- and orcokinin-immunoreactivity (Soehler et al. 2011). According to
this rule, all large PDFMEs, found at anterior or posterior position, do not coexpress FMRF- and
orcokinin-immunoreactivity and the sum of these neurons was shown to be always six. These
neurons were suggested to be a group with a common developmental origin, from which some can
move to a posterior position during development. Alternatively, all six large somata can be found at
anterior position (Soehler et al. 2011).
PDF-ir arborizations in the AME were found mainly in the interglomerular and shell neuropil and
always contained large and medium-sized DCVs (Reischig and Stengl 1996). The large PDFMEs were
shown to contain medium-sized DCVs and have synapses with fibers in the anterior and shell
neuropil of the AME (Reischig and Stengl 2003b). In contrast to large PDFMEs, medium-sized
aPDFMEs contain large DCVs and innervate mainly the interglomerular neuropil but also the anterior
neuropil of the AME. Some of the PDFMEs were shown to coexpress different neuropeptides: All
small and medium-sized aPDFMEs and the small pPDFMEs always coexpress FMRFamideimmunoreactivity. All medium-sized aPDFMEs and a subgroup of the small aPDFMEs, but not the
pPDFMEs, also coexpress orcokinin- and baratin-like immunoreactivity (Soehler et al. 2011). One
neuron of the medium-sized aPDFMEs additionally shows MIP-immunoreactivity and thus could
possibly coexpress five neuropeptides: baratin, FMRFamide, MIP, orcokinin and PDF (Soehler et al.
2011; Schulze et al. 2012). Remarkably, the medium-sized aPDFMEs were the first neurons reported
to express more than two neuropeptides in a single neuron (Soehler et al. 2011). While the somata
showed immunolabeling for PDF, FMRFamide and orcokinin, double-labeling in fibers and terminals
for PDF and orcokinin was never observed and just a few termination sites but no commissural fibers
showed double-labeling for PDF and FMRFamide. It was suggested that these neurons employ
28
Introduction
peptide sorting and that the differential release of neuropeptide mixtures could be used to phasecontrol specific physiological processes daytime dependently (Soehler et al. 2011).
Fig. 12. Neurons associated with the accessory medulla of R. maderae. A. Three-dimensional model of the accessory
medulla (AME) with associated neurons, redrawn and modified after (Reischig and Stengl 2003b). The distal tract (DT) is
suggested to transmit light input into the AME B. Scheme of the localization of PDF-ir medulla neurons (PDFMEs), modified
from (Soehler et al. 2011). The number of the grey circles does not reflect the correct sizes of the groups. Three mediumsized and the largest anterior PDFMEs, together with another VNe, are suggested to form a coupling pathway to the
contralateral AME. For further details see text. Abbreviations: ANe: anterior neurons, di: distal, do: dorsal, DFVNe: distal
frontoventral neurons, MFVNe: medial frontoventral neurons, MNe: medial neurons, VNe: ventral neurons, VPNe:
ventroposterior neurons. Scale bar: 50 µm.
The branching pattern of the PDFLAs appears to be restricted to the optic lobes, where the lamina,
the distal medulla and the AME are innervated (Stengl 1994; Petri et al. 1995; Reischig et al. 2004;
Wei et al. 2010). In the optic lobe the projections of the PDFMEs connect the AME to the medulla
and the lamina via the anterior fiber fan and/or the median layer fiber system of the medulla (Fig. 13,
Fig. 14). The fibers of the anterior fiber fan were shown to innervate the distalmost layer of the
medulla and the proximal layer of the lamina. On the other hand the PDFMEs project to different
targets in the protocerebrum and the contralateral optic lobe via the anterior optic commissure and
the posterior optic commissure. These fibers leave the optic lobe via the lobula valley tract, which
runs from the proximal end of the medulla to the proximal end of the lobula. There the fibers
bifurcate into the AOC and the posterior optic tract, which leads into the POC (Stengl 1994; Stengl
and Homberg 1994; Reischig and Stengl 2002, 2003b; Wei et al. 2010). Several branching sites,
termed plexi (p1 - p5) and anterior fiber plexus (AFP), and two meeting points of fiber bundles,
termed arborization areas (a1 and a2), can be detected (Fig. 13, Fig. 14). The fibers of the AOC
innervate the SLP and SMP, branch in the plexi p3 - p5, and show the arborization areas a1 and a2.
The fibers running through the POC extensively branch in p1, located within the LOVT, and p2 and
innervate the POTU. Remarkably, two plexi, p1 and AFP, were shown to connect several PDF-ir
branching sites: Fibers originating from p1 directly interconnect the AME with the plexi p2, p3, and
AFP and with the SLP, ILP, and VLP. Fibers originating from the AFP, which is located laterally to the
mushroom body, interconnect the AME with all plexi, the arborization area a1 and the SLP. Thus,
29
Introduction
these plexi connect the AOC with the POC, allowing for exchange of information between fibers
running through both commissures (Wei et al. 2010). Additionally, PDF-ir ramifications from an
unknown soma were detected posterior to the lobus glomerulatus in the tritocerebrum (Fig. 13,
Fig. 14).
Fig. 13. Branching pattern of the PDF expressing neurons in the brain of R. maderae. PDF-ir neurons were implemented
in a three-dimensional model of R. maderae's brain. The brain is shown in frontal view. The PDF-ir neurons innervate the
optic lobes and different areas of the protocerebrum. Additionally, the antennal lobe (AL) in the deutocerebrum and the
lobus glomerulatus (LG) in the tritocerebrum are innervated. For further information see text. Abbreviations: AME:
accessory medulla, AOC/POC: anterior/posterior optic commissure, CA: calyx, CBL/CBU: lower/upper division of the central
body, LA: lamina, LO: lobula, MB: mushroom body, ME: medulla, ML: median lobe, PB: protocerebral bridge, PED:
pedunculus, VL: ventral lobe. Scale bar: 300 µm. Modified after (Wei et al. 2010).
Fig. 14. Arborization sites of the PDF-expressing neurons. The PDF-immunoreactive medulla neurons (PDFMEs) arborize in
all optic lobe neuropils and in different arborization sites (a1 and a2) and plexi (AFP and p1 - p5) in the protocerebrum. For
further information see text. Abbreviations: AFP: anterior fiber plexus, AL: antennal lobe, AME: accessory medulla, AN:
antennal nerve, aPDFME: anterior PDFME, AOC/POC: anterior/posterior optic commissure, CX: central complex, ILP:
inferior lateral protocerebrum, LA: lamina, LO: lobula, MB: mushroom body, ME: medulla, ON: optic nerves, SLP: superior
lateral protocerebrum, SMP: superior median protocerebrum. Modified after (Wei et al. 2010).
30
Introduction
Indicated by varicosities, PDF was suggested to be released in the lamina, the AME, all five plexi, the
arborization areas a1 and a2, the POTU, SLP, and SMP. Interestingly, the distances between
arborization sites of PDF-ir fibers in the AME, all five plexi, both arborization areas, and the POTU
were shown to be not significantly different from each other or integer multiples from each other. It
was suggested, that the relationship between the distances could contribute to different delay lines,
possibly enabling postsynaptic neurons to measure phase differences (Wei et al. 2010).
Two of the three presumptive AMAE coupling tracts (tract 4 and 7) resembled the characteristic
branching pattern of the PDFMEs (Reischig and Stengl 2002). Indeed, the combination of the tracer
backfill/injection strategy with immunocytochemistry revealed that four aPDFMEs form a direct
coupling pathway for both AMAE (Reischig et al. 2004; Soehler et al. 2011). Further
immunocytochemical double- and triple-labeling studies demonstrated that orcokinin and
FMRFamide also play a role in coupling, and that PDF, orcokinin, and FMRFamide are partially
colocalized in the coupling neurons (Hofer and Homberg 2006a; Soehler et al. 2011). Four aPDFMEs
(the largest and three medium-sized somata), belonging to the VNe cells, were shown to form the
MC I group. The largest aPDFME did not coexpress orcokinin or FMRFamide and was shown to
project via the POC and the AOC to the contralateral AME and to most if not all midbrain targets in
both brain hemispheres. In contrast, the three medium-sized aPDFMEs coexpress PDF, orcokinin,
FMRFamide, and baratin. These cells appear to project solely via the AOC and innervate at least the
contralateral AME, the POTU and the dorsal part of the SLP (Soehler et al. 2011).
PDF was also detected via mass spectrometry in brains of R. maderae. The peptide sequence could
be identified except for one amino acid, whose identity was leucine or isoleucine (Hamasaka et al.
2005). Recently, the identity of isoleucin4 was confirmed in a transcriptome analysis (personal
communication with Achim Werckenthin, University of Kassel). Thus, the sequence of RhyparobiaPDF is identical to Meimuna opalifera-, A. domesticus-, and G. bimaculatus-PDF (Tab. 3).
Tab. 3. PDF sequences of different insect species (modified from (Hamasaka et al. 2005))
Diptera
Anopheles gambiae
Musca domestica
Phormia regina
Hemiptera
Meimuna opalifera
Orthopteromorpha
Acheta domesticus
Carausius morosus
Gryllus bimaculatus
Periplaneta americana
Rhyparobia maderae
Romalea microptera
NSELINSLLSLPKTMNDAa
NSELINSLLSLPKNMNDAa
NSELINSLLSLPKSMNDAa
NSELINSLLSLPKNMNDAa
(Matsushima et al. 2003)
(Park and Hall 1998)
NSEIINSLLGLPKVLNDAa
(Sato et al. 2002)
NSEIINSLLGLPKVLNDAa
NSELINSLLALPKVLNDAa
NSEIINSLLGLPKVLNDAa
NSELINSLLGLPKVLNDAa
NSEIINSLLGLPKVLNDAa
(Rao and Riehm 1988)
(mentioned in Hamasaka et al. 2005)
(Singaravel et al. 2003)
(mentioned in Hamasaka et al. 2005)
(Hamasaka et al. 2005), personal communication
with Dr. Achim Werckenthin (University of Kassel)
(Rao et al. 1987)
NSEIINSLLGLPKLLNDAa
The variable amino acids at positions 4, 10, 14, and 15 are color-coded.
To date it is still not fully understood, whether PDF is only a non-photic input and also an output
signal of the circadian clock, or whether it also plays a role in the light entrainment pathway. For
31
Introduction
injections of synthetic Arg13-A. domesticus PDF into the vicinity of the AME, a monophasic all-delay
PRC with maximum phase-delays at the late subjective day was obtained (Petri and Stengl 1997).
Because the PRC was not light-like, it was suggested that PDF functions as non-photic input signal
(Petri and Stengl 1997) and as coupling signal from the contralateral AME (Reischig and Stengl 2002;
Reischig et al. 2004). However, more recent injections of P. americana-PDF into the complex eye
resulted in a biphasic, light-like PRC suggesting that PDF could function in light entrainment
(Schendzielorz et al. 2014). The non-photic function of PDF was also supported by intracellular
recordings, in which AME-neurons, which resembled parts of the PDF-ir branching pattern, were
shown to be insensitive to light during the daytime (Loesel and Homberg 2001). However, there is
evidence for light-sensitivity of PDF-ir neurons: When cockroaches were raised in non-24 h periods
(T22 = LD 11:11, T26 = LD 13:13) or in different photoperiods (LD 6:18 or LD 18:6) the number and
the branching pattern of PDFMEs was affected (Wei and Stengl 2011). The medium-sized aPDFMEs
were shown to be most light-responsive in these experiments: The number of somata increased with
increasing period length and increasing photoperiod, the number of PDF-ir fibers in the AOC
increased with increasing period length, and the length of the fibers in the AOC and POC increased
with longer photoperiods. Therefore, it was hypothesized that the medium-sized aPDFMEs could
have longer endogenous periods, enabling them to couple better to longer exogenous periods, and
that these neurons are activated by light and inhibited by darkness only at ZT 11-13 (Wei and Stengl
2011).
In another cockroach species, B. germanica, pdf expression was knocked down via RNAi with an
astonishing efficiency: Starting from the second day after injection of double-stranded pdf RNA, pdf
mRNA levels decreased and the animals lost their locomotor activity rhythm in DD as well as LD
conditions and also decreased their amount of activity, demonstrating the importance of PDF
signaling in this insect species (Lee et al. 2009).
1.4.7.2 Other neuropeptides and transmitters of the AME
Around 25 neurons in the vicinity of the AME show GABA-immunoreactivity (Tab. 4). These neurons
belong to different groups (DFVNe, MFVNe, MNe, VMNe, and VNe) and innervate mainly the
glomeruli of the AME. Some of them project via the distal tract, the presumptive ipsilateral photic
entrainment pathway, to different layers of the medulla and lamina. Additionally, GABA-ir MNes,
which connect the glomeruli of the AME with different layers of the medulla, accessory laminae, and
the proximal lamina, might transmit ipsilateral light information (Petri et al. 2002; Schendzielorz and
Stengl 2014). Thus, GABA-ir neurons were suggested to play a role in photic entrainment. Indeed, the
PRC obtained for GABA injections in the vicinity of the AME resembles the typical biphasic shape with
phase-delays in the early subjective night and phase-advances in the late subjective night, which can
also be obtained for light pulses (Page and Barrett 1989; Petri et al. 2002, Fig. 2). Moreover, neurons,
which connected the AME to the medulla and the lamina and resembled the morphology of GABA-ir
AME neurons, responded in intracellular recordings to light stimuli (Loesel and Homberg 2001).
Expression of ACh could be demonstrated indirectly by the presence of acetylcholinesterase (AChE,
the enzyme degrading ACh), which was detected histochemically in the glomeruli of the AME and all
other optic lobe neuropils (Tab. 4). However, this method did not allow the staining of somata in the
optic lobe (Schendzielorz 2013). Calcium imaging experiments, performed on AME cells in vitro,
32
Introduction
demonstrated widespread receptivity to ACh for nearly all AME cells (96 %). ACh application resulted
in an increase of [Ca2+]i, typically with subsequent oscillations, which is mediated by nicotinic ACh
receptors (Baz et al. 2013). Thus, ACh appears to provide a general excitatory input to the AME.
Injections of ACh into the cockroach's hemolymph resulted in an all-delay PRC with maximum delay
at the late subjective day, probably indicating that ACh gates an inhibitory pathway at dusk
(Schendzielorz 2013).
Histamine-immunostaining was detected in ventral parts of the loose neuropil of the AME (Tab. 4).
The fibers were shown to originate from one centrifugal neuron with soma in the posterior lateral
protocerebrum and arborizations in the anterior optic tubercle (AOTU), the ILP, the lateral horn (LH),
a proximal layer of the medulla, and the AME. Varicosities of the stained terminals in the AME
suggested that the AME is an output region of this neuron (Loesel and Homberg 1999). Interestingly,
there is no innervation of the AME by the histaminergic photoreceptors of the compound eye and
thus no direct photic input (Loesel and Homberg 1999). In calcium imaging experiments around half
of the tested AME cells (48,65 %) responded to histamine, either showing decreases of [Ca2+]i or
irregular changes in spontaneous calcium oscillations. Apparently these effects were mediated by
histamine-gated chloride channels, which could be blocked by the mammalian type 2 histamine
receptor (H2) antagonist cimetidin but not by the chloride channel blocker picrotoxin (PTX, Baz et al.
2013). Thus, histamine provides non-photic, inhibitory input to a subpopulation of AME cells.
Serotonin-immunoreactivity was found in 57 - 99 lamina cells, which largely coexpressed PDF, and 18
- 25 anterior medulla cells with processes in the interglomerular and shell neuropil of the AME (Petri
et al. 1995, Tab. 4). Among these neurons are projection neurons of the AME, which send fibers into
the midbrain and/or the lamina, and tangential neurons of the medulla, which send sidebranches
into the AME and connect AME and medulla (Petri et al. 1995). Serotonin-infusion for several hours
in the head capsule resulted in an all-delay PRC with maximum phase-delays in the late subjective
day and the early subjective night (Page 1987).
Allatostatin-immunoreactivity was found in 16 - 21 anterior medulla neurons, which mainly innervate
the interglomerular and shell neuropil of the AME (Tab. 4). Among these cells are tangential neurons
connecting the AME with medial layers of the medulla and neurons, which send fibers towards the
superior protocerebrum (Petri et al. 1995; Homberg et al. 2003).
Up to 31 AME neurons express allatotropin-immunoreactivity (Tab. 4). Most of these neurons are
local interneurons of the AME, belonging to the DFVNe and MFVNe group, but among the VNes and
MNes allatotropin-ir neurons are found, too. Allatotropin-ir arborizations in the AME can be found
mainly in the glomerular neuropil (Petri et al. 1995; Homberg et al. 2003; Reischig and Stengl 2003b;
Hofer and Homberg 2006b). Allatotropin-ir MNes, which connect the glomeruli of the AME with
different layers of the medulla, accessory laminae, and the proximal lamina, were assumed to
transmit ipsilateral light information (Schendzielorz and Stengl 2014). Consistently, injection of
Manduca sexta-allatotropin (Mas-allatotropin) in the vicinity of the AME resulted in a biphasic, lightlike PRC, suggesting that allatotropin plays a role in light entrainment (Petri et al. 2002). However,
recently Rhyparobia-allatotropin was identified via mass spectrometry and injection of this peptide in
the head capsule did not result in a biphasic but instead in a monophasic all-delay PRC with
maximum delay at the late subjective day (Schulze et al. 2013).
33
Introduction
One cluster of around ten baratin-ir neurons, which densely innervate the AME, has been detected
and termed "ol3", but the detailed projections of these neurons were not characterized (Nässel et al.
2000). However, apparently all medium-sized aPDFMEs and some of the small aPDFMEs coexpress
baratin-immunoreactivity (Soehler et al. 2011). Thus, the baratin-ir neurons of the ol3-cluster
apparently belong to the VNe and DFVNe cluster (Tab. 4).
In contrast to all other neuropeptides, corazonin-immunoreactivity was found only in one anterior
medulla neuron (the cell group has not been specified), which arborizes in the interglomerular and
shell neuropil of the AME and connects the AME to medial layers of the medulla (Petri et al. 1995,
Tab. 4).
Several extended FMRFamides, among them AVRDNFIRFamide and leucomyosuppressin (LMS), and
short neuropeptide F (sNPF) could be detected in the AME by mass spectrometry (Soehler et al.
2008, Tab. 4). FMRF-immunoreactivity is expressed in around 24 AME neurons (Petri et al. 1995;
Soehler et al. 2008). These neurons belong to the VNe, DFVNe, MNe, VPNe, and ANe cluster and thus
apparently to different functional circuits of the AME. In the AME FMRFamide-ir fibers were found in
the anterior and interglomerular neuropil, but also in the glomeruli. FMRFamide-ir projections were
shown in all optic lobe neuropils. The FMRFamide-ir fan-shaped anterior fiber system connected the
AME with the medulla and the lamina. In the midbrain FMRFamide-ir projections are found in the
SMP, SLP, ILP, and VLP. However, next to the 24 FMRFamide-ir AME neurons, around 80 additional
neurons can be found in the optic lobe and 1100 in the central brain displaying FMRFamideimmunoreactivity. Therefore, it is difficult to assign FMRFamide-ir projections to distinct neurons.
However, in triple labeling experiments it was shown, that three FMRFamide-ir VNes (identical to
three AMAE-coupling, medium-sized aPDFMEs) belong to the MC I group and project via the AOC to
the contralateral AME. No FMRFamide-ir neurons were detected among the other medulla cell
groups, which were shown to project into the contralateral optic lobe (MC II - IV, Soehler et al. 2011).
While the anti-FMRFamide antibodies employed in these studies were suggested to detect most or
all FMRFamide related peptides (FaRPs), antibodies against drosomyosuppressin (DMS) or sNPF were
shown to be more specific. With both antibodies a subpopulation of the FMRFamide-ir neurons was
labeled (Soehler et al. 2007; Soehler et al. 2008). Two VNes and one ANe were shown to be sNPF-ir.
These neurons had arborizations in the interglomerular and glomerular neuropil of the AME, the
anterior fiber fan and the middle layer of the medulla, the lamina, and in the SMP, SLP, ILP, and VLP
(Soehler et al. 2008). Up to three neurons, belonging to the VNes and ANes, were shown to be
DMS-ir and thus were suggested to express LMS. Probably these neurons are restricted to the
ipsilateral optic lobe and arborize mainly in the interglomerular and anterior shell neuropil of the
AME and project via the fan-shaped anterior layer fiber system of the medulla to the accessory
laminae and to proximal medulla layers (Soehler et al. 2007). The morphology of DMS-ir VNes
resembled the light-sensitive OL2 neurons, which were suggested to deliver excitatory light input to
the AME (Loesel and Homberg 2001). However, injection of LMS in the vicinity of the AME did not
affect the phase of circadian locomotor activity, arguing against a role of LMS in light entrainment
(Soehler et al. 2007). In contrast, injections of FMRFamide or P. americana-FMRFa-7 (Pea-FMRFa-7,
= DRSDNFIRFamide) had prominent effects (Soehler et al. 2008) on the phase of locomotor activity.
FMRFamide injections resulted in dose-dependent phase-shifts: the higher concentration (100 fmol)
resulted in phase-delays at CT 8 and CT 18 and the lower concentration (10-2 fmol) resulted in a
phase-advance at CT 18. It was suggested that the comparatively unspecific FMRFamide activated
different FaRP receptors and thus, the FMRFamide PRC represents an overlay of PRCs for different
34
Introduction
FaRPs. In contrast, for Pea-FMRFa-7 injections an all-delay PRC with maximum phase-delay at CT 4
was obtained (Soehler et al. 2008).
One cluster of up to ten neurons located anterioventrally to the AME was found to express
gastrin/cholecystokinin-immunoreactivity (Tab. 4). These neurons sparsely innervate the AME
without preference for a special part of the neuropil (Petri et al. 1995).
Up to 18 AME neurons were shown to be leucokinin 1-ir (Tab. 4). The somata are located at a
position anterioventrally to the AME. These neurons are tangential neurons, which connect the AME
with the distalmost layer of the medulla and innervate mainly the central glomeruli but also the shell
of the AME (Petri et al. 1995).
With mass spectrometric methods five different MIPs were detected in the AME (Lem-MIP-1 - 5,
Schulze et al. 2012, Tab. 4). MIP-immunostainings showed around 30 labeled somata, distributed in
all AME-associated cell groups except for the ANes. MIP-immunoreactivity was detected in the
glomeruli as well as the interglomerular and shell neuropil of the AME, suggesting that MIPs play
different roles in the AME. Besides the AME, MIP-immunoreactivity can be detected particularly in
the SMP, parts of the central complex and the tritocerebrum, but the high number of MIP-ir neurons
(around 700 somata in both optic lobes and around 1560 somata in the central brain) does not allow
the exact assignment of arborizations to the AME neurons. However, some of the MIP-ir fibers
crossing the midbrain via the POC could be assigned to six MIP-ir somata of the VMNe group. These
cells are identical with the MC II group, which projects to the contralateral optic lobe. Moreover,
colocalization of MIP- and PDF-immunoreactivity was detected in one large and one medium-sized
aPDFME belonging to the VNes. Double-labeled fibers in the fan-shaped anterior-layer fiber system
and the distalmost layer of the medulla, in the proximal lamina, the posterior first optic chiasm, the
POTU, and the POC could be assigned to these neurons (Schulze et al. 2012). The three medium-sized
aPDFMEs, which couple both AMAE, were shown to project via the AOC to the contralateral side
(Soehler et al. 2011), but no colocalization of MIP- and PDF immunoreactivity was found in the AOC.
Thus, the medium-sized aPDFME that colocalizes PDF- and MIP-immunoreactivity either does not
belong to the three medium-sized aPDFMEs coupling both AMAE, or peptide sorting is employed as
described before (Soehler et al. 2011). Among the large aPDFMEs only the largest has been shown to
couple the contralateral AME via the POC and the AOC (Soehler et al. 2011). However, the largest
aPDFME does not coexpress MIP-immunoreactivity. Instead another large aPDFME, possibly without
coupling function, colocalizes MIP- and PDF-immunoreactivity and seems to project via the POC to
the ipsilateral and possibly the contralateral POTU (Schulze et al. 2012). Additionally, one VMNe and
one VNe was shown to colocalize MIP- and orcokinin-immunoreactivity (Schendzielorz and Stengl
2014). MIP-ir MNes, which connect the glomeruli of the AME with different layers of the medulla,
accessory laminae, and the proximal lamina, were assumed to provide ipsilateral light information
(Schendzielorz and Stengl 2014). Injections of Rhyparobia-MIP-1 into the head capsule resulted in a
monophasic all-delay PRC with maximum phase-delay in the early subjective night (Schulze et al.
2013), while injections of Rhyparobia-MIP-2 resulted in an all-advance PRC with maximum phaseadvance in the late subjective night (Schendzielorz and Stengl 2014).
35
Introduction
Tab. 4. Neuropeptides and neurotransmitters of AME neurons
ACh
# neurons
?
GABA
25
Histamine
1
Serotonin
18-25
Allatostatin
16-21
Allatotropin
31
Baratin
10
Corazonin
1
FMRFamides
24
LMS
3
sNPF
3
Gastrin/
Cholecystokinin
Leucokinin
10
MIPs
30
Orcokinin
30
PDF
16/50-70
18
Remarks
96 % of AME cells responded excitatory to ACh in
calcium imaging experiments; AChE-immunoreactivity in
the glomeruli of the AME; all-delay PRC.
Belong to the DFVNe, MFVNe, MNe, VMNe, and VNe
group; inhibition of AME neurons in 98 % of the
experiments; biphasic light-like PRC; role in photic
entrainment and synchronization.
Soma located in the posterior lateral protocerebrum;
innervation of the loose neuropil of the AME; around 50
% of the AME cells responded to histamine in calcium
imaging experiments.
Innervation of interglomerular and shell neuropil of the
AME; all-delay PRC.
Innervation of the interglomerular and shell neuropil of
the AME.
Belong to the DFVNe, MFVNe, VNe and MNe cluster;
innervation of the glomerular neuropil of the AME;
biphasic light-like PRC for injection of Mas-allatotropin;
all-delay PRC for injection of Rhyparobia-allatotropin.
Neurons of the ol3-cluster; belong partially to the VNe
and DFVNe groups (all medium-sized aPDFMEs and
some small aPDFMEs).
One anterior medulla neuron; innervation of
interglomerular and shell neuropil of the AME.
Belong to the VNe, DFVNe, MNe, VPNe, and ANe group;
innervation of anterior, glomerular and interglomerular
neuropil of the AME; three FMRFamide-ir VNes couple
both AMAE (= medium-sized aPDFMEs, belong to MC I
group); dose-dependent phase-delays or -advances.
Belong to the VNe and ANe group; innervation of the
anterior and interglomerular AME neuropil; no LMSdependent phase-shifts.
Belong to the VNe and ANe group; innervation of the
glomerular and interglomerular AME neuropil;
Somata located anterioventrally to the AME.
Reference
Belong to the DFVNe group; innervation of glomeruli and
shell of the AME.
Belong to DFVNe, MFVNe, MNe, VNe, VMNe, and VPNe
group; innervation of the glomerular, interglomerular,
and shell neuropil of the AME; six MIP-ir VMNes (MC II)
are involved in coupling; all-delay PRC; five different
MIPs were detected.
Belong to the DFVNe, VPNe, VNe, VMNe, and MNe
group; innervation of all AME neuropils; three orcokininir VMNes (MC II) and three VNes (= medium-sized
aPDFMEs, belonging to MC I) are involved in coupling;
two different orcokinins were detected; biphasic lightlike PRCs, partially light-sensitive.
16 PDFMEs (belonging to the VNe and DFVNe group) and
50-70 PDFLAs; best characterized AME neurons; for
further information see text.
(Petri et al. 1995; Hofer and
Homberg 2006b)
(Baz et al. 2013; Schendzielorz
2013)
(Petri et al. 2002; Schneider and
Stengl 2005; Schendzielorz and
Stengl 2014)
(Loesel and Homberg 1999; Baz et
al. 2013)
(Page 1987; Petri et al. 1995)
(Petri et al. 1995; Homberg et al.
2003)
(Petri et al. 1995; Petri et al. 2002;
Homberg et al. 2003; Reischig and
Stengl 2003b; Hofer and Homberg
2006b; Schulze et al. 2013;
Schendzielorz and Stengl 2014)
(Nässel et al. 2000; Soehler et al.
2011)
(Petri et al. 1995)
(Petri et al. 1995; Soehler et al.
2008; Soehler et al. 2011)
(Soehler et al. 2007)
(Soehler et al. 2008)
(Petri et al. 1995)
(Schulze et al. 2012; Schulze et al.
2013)
(Hofer and Homberg 2006b, a;
Soehler et al. 2011; Wei and
Stengl 2011)
(Homberg et al. 1991; Stengl and
Homberg 1994; Petri et al. 1995;
Reischig and Stengl 1996, 2003b;
Reischig et al. 2004; Hamasaka et
al. 2005; Wei et al. 2010; Soehler
et al. 2011; Wei and Stengl 2011)
Furthermore, widespread orcokinin-immunoreactivity can be found in the cockroach brain,
originating from around 1400 somata, of which 30 were shown to innervate the AME (Hofer and
Homberg 2006b, a; Soehler et al. 2011). These neurons have arborizations in the interglomerular, the
36
Introduction
distal and anterior shell neuropil, and to a lesser extent in the glomeruli. The somata can be found
among the DFVNe, VPNe, VNe, VMNe, and MNe group (Hofer and Homberg 2006b; Soehler et al.
2011; Schendzielorz and Stengl 2014, Tab. 4). The projection pattern of three orcokinin-ir neurons
among the VMNes (= MC II), one of which colocalized MIP-immunoreactivity, could be reconstructed,
showing arborizations in median layers of the medulla, the interglomerular neuropil of the AME, and
fibers running through the LVT and the POC to the contralateral AME and medulla but not into the
anterior fiber fan (Hofer and Homberg 2006b, a). Interestingly, these neurons resembled the
projection pattern of polarized light-sensitive PC2-neurons (Loesel and Homberg 2001) and were
suggested to contribute to contralateral light entrainment. Additionally, orcokinin-ir MNes, which
connect the glomeruli of the AME with different layers of the medulla, accessory laminae, and the
proximal lamina, might transmit ipsilateral light information (Schendzielorz and Stengl 2014). Next to
the three neurons of the MC II group, three orcokinin-ir VNes (identical with three of the mediumsized aPDFMEs) were shown to couple both AMAE via the AOC and thus belong to the MC I group
(Soehler et al. 2011). Colocalization with other neuropeptides or neurotransmitters was detected in
different VNe cells (PDF-, FMRF-, GABA-, Mas-allatotropin-, and MIP-immunoreactivity), one MNe
(Mas-allatotropin-immunoreactivity), and one VMNe (MIP-immunoreactivity, Hofer and Homberg
2006b; Soehler et al. 2011; Schendzielorz and Stengl 2014). Two different orcokinins were mass
spectrometrically identified in the AME: Rhyparobia-ORC-1 and -2 (Schulze et al. 2013). Injection of
these peptides resulted in a light-like, biphasic PRC (Schulze et al. 2013) as shown before for
injections of the crustacean Asn13-orcokinin (Hofer and Homberg 2006a), indicating a role in light
entrainment. Moreover, orcokinin-ir neurons appear to be light sensitive, since the number of
orcokinin-ir fibers in the AOC (most probably originating from orcokinin-ir, medium-sized aPDFMEs)
and the middle fiber bundle of the POC (probably originating from the orcokinin-ir VMNes, belonging
to the MC II cells) were shown to increase with increasing photoperiod (Wei and Stengl 2011).
1.4.8 Clock genes
In contrast to the fruit fly D. melanogaster, there is not much information available on the molecular
circadian clockwork of cockroaches, possibly because no cockroach genome is available and because
of limited facilities for genetic manipulations. Just a few cockroach clock genes have been cloned so
far. For the American cockroach Periplaneta americana only partial sequences of per (Reppert et al.
1994) and tim (Sehadova et al., unpublished) are known and for two Blattella species, the German
cockroach B. germanica (Lin et al. 2002a) and the double-striped cockroach B. bisignata (Yang et al.,
unpublished), only per-sequences are known. Only recently per, cry2, and a partial sequence of tim of
Rhyparobia maderae were cloned and analyzed (Werckenthin et al. 2012). The expression level of all
three genes was shown to cycle in brains and isolated AMAE in a daytime-dependent manner with
expression peaks in the first half of the night. For tim and cry2, but not for per, the cycling continued
in constant darkness. Even though R. maderae is an equatorial species, in which no photoperiodic
behavior has been found, the expression levels of per, tim and cry2 were shown to adjust to different
photoperiods with peaks at the beginning of the scotophase (Werckenthin et al. 2012).
While D. melanogaster expresses only cry1, which primarily functions as a blue-light photoreceptor in
the central pacemaker, in R. maderae only the mammalian-type cry2, but not the Drosophila-type
cry1, was found so far, as is the case for the honeybee Apis mellifera and the red flour beetle
37
Introduction
Tribolium castaneum. Other insects, like the monarch butterfly Danaus plexippus or the mosquito
Anopheles gambiae, express both types of cryptochrome. When expressed in Drosophila Schneider 2
(S2) cells, the Drosophila-type CRY1 proteins of these species were shown to be light-dependently
degraded, while the mammalian-type CRY2 proteins were shown to be light-insensitive and to
repress CLOCK-CYCLE-mediated transcription, similar to Mus musculus mCRY1 (a type-2 CRY, Zhu et
al. 2005). Even if no functional studies were performed, it seems likely that, R. maderae CRY2 also
functions as transcriptional repressor instead of being a blue-light photoreceptor. Consistently, it
cycles in phase with per and tim, which is not the case for cry1 in D. melanogaster. In addition,
R. maderae PER shows a conserved C-terminal c7 domain, which is not present in D. melanogaster
PER and mediates binding of a type-2 CRY in mammals (Werckenthin et al. 2012). In this respect the
clockwork of R. maderae is more similar to the clockwork of mammals than to the clockwork of
Drosophila.
To date it is largely unknown, in which neurons in the brain of R. maderae different clock genes are
expressed. Preliminary immunostainings with antibodies directed against PER sequences of different
insect species, among them R. maderae PER, showed widespread expression of per. Not only
neurons associated with the AME but virtually all neurons and apparently glial cells of the brain and
all other ganglia showed always nuclear and never cytoplasmatic PER-like immunoreactivity,
suggesting a more general function of PER in R. maderae (Werckenthin 2013). For other clock genes
than per, no data are available, neither on the protein nor on the mRNA level.
1.4.9 Model of the AME
In conclusion, the AME is densely innervated by about 300 neurons, which virtually all express
nuclear PER-immunoreactivity (Werckenthin 2013). However, because of restricted knowledge about
the molecular circadian clockwork and the identity of the clock gene expressing neurons, previous
studies concentrated mainly on the neuropeptide or neurotransmitter content of the neurons.
Among the AME associated neurons different functional cell types like local neurons, light-input
neurons, projection (output) neurons, or coupling neurons can be found and several neurons serve
more than one function. Coupling of both AMAE is achieved by FMRFamide-ir, MIP-ir, PDF-ir,
orcokinin-ir, and other so far uncharacterized neurons of the MC I group (subpopulation of the VNe
group), the MC II group (identical to the VMNe group), and probably also by neurons of the MC III
and MC IV groups. While MC II cells were suggested to pass contralateral light information, MC I cells
were suggested to pass synchronizing phase information to the anterior and shell neuropil of the
AME (Reischig and Stengl 2002; Reischig et al. 2004; Hofer and Homberg 2006b, a). Output neurons
can be found among the VNes but also among other cell groups, showing immunoreactivity for
allatostatin, allatotropin, FMRFamide, orcokinin and PDF. These neurons innervate mainly the
interglomerular and shell neuropil of the AME and pass information to the lamina and to different
targets in the protocerebrum, indicated by varicosities (Homberg et al. 2003). The AME only receives
indirect light input from the compound eyes, since the terminals of histaminergic photoreceptors
were only found in the lamina and distal medulla but not in the AME (Loesel and Homberg 1999).
Apparently, light input from the ipsilateral compound eye is transmitted by GABAergic neurons,
which connect the lamina and medulla with the glomerular neuropil of the AME via the distal tract.
Additionally, ipsilateral light information might be transmitted by allatotropin-, GABA-, MIP-, and
38
Introduction
orcokinin-ir neurons of the MNe group, which connect the glomeruli of the AME with different layers
of the medulla, the accessory laminae, and the proximal lamina (Petri et al. 2002; Schendzielorz and
Stengl 2014). Contralateral light input appears to be mediated by three orcokinin-ir VMNes (one of
which also expresses MIP-immunoreactivity), which innervate mainly the interglomerular neuropil of
the AME and project via the POC to the contralateral optic lobe, and one VNe coexpressing MIP and
orcokinin, which projects via the AOC to the contralateral optic lobe (Hofer and Homberg 2006b, a;
Schendzielorz and Stengl 2014). Local neurons of the AME can be found among the DFVNes and
MFVNes and were shown to be allatostatin-, allatotropin-, FMRFamide-, orcokinin-, and PDF-ir (Petri
et al. 1995; Reischig and Stengl 2003b; Hofer and Homberg 2006b, a; Soehler et al. 2008). These
neurons arborize mainly in the glomerular neuropil of the AME and are involved in local network
interactions. Thus, the subcompartments of the AME apparently serve different functions: The
glomerular core is involved in processing of ipsilateral light input and other network interactions,
while the interglomerular, the anterior, and the shell neuropil are involved in the transmission of
efferent output and processing coupling input (Homberg et al. 2003).
1.4.10 Physiological characterization of the accessory medulla
neurons
1.4.10.1
AME neurons are coupled to synchronous spiking assemblies
Schneider and Stengl developed a method for long-term extracellular recordings from punched out
AMAE with associated neurons, allowing the measurement of electrical activity for several hours up
to several days (Schneider and Stengl 2005, 2006, 2007). In these multi-unit-recordings the temporal
coordination of electrical activity from several neurons of the network was investigated. In more
than 80 % of the experiments the AME-neurons were shown to fire very regular APs, which were
sensitive to TTX (Schneider and Stengl 2005). Apparently, these regularly active cells are ultradian
oscillators, whose membrane potential oscillates in the timescale of milliseconds (ultradian period =
interevent-interval). Interestingly, the spontaneous AP activity often lies in a frequency range
reminiscent of gamma-band oscillations (20 - 100 Hz), which subserve important functions in the
mammalian cortex, such as perceptual binding and formation of functional assemblies (Reviews:
Singer and Gray 1995; Buzsáki and Wang 2012; Khazipov et al. 2013; Merker 2013). In the AME the
neurons are also coupled to synchronous spiking assemblies by synaptic and non-synaptic (within the
meaning of "non-chemical-synaptic") interactions. Within one assembly the cells fire with the same
or integer multiples of the same frequency (with harmonic frequencies) and at the same phase
(= timing of the spikes). Cells of different assemblies fire with harmonic frequencies but with a stable
phase difference (Schneider and Stengl 2005). In the majority of experiments (71 %) interruption of
synaptic transmission resulted in a disinhibition of activity and splitting of assemblies. Assemblies,
which were previously synchronized to the same phase ("phase-locked"), now fired with a stable
phase difference (Schneider and Stengl 2005, 2006). This effect could be mimicked by PTX, which also
blocks GABAA-receptors. Additionally, GABA was shown to phase-lock different assemblies before the
activity was entirely inhibited. Thus, it was concluded that inhibitory synaptic interactions, apparently
mainly GABAergic interactions, also indicated by prominent GABA-immunoreactivity in the AME,
form assemblies of synchronized and phase-locked cells (Schneider and Stengl 2005). With the use of
39
Introduction
different gap junction blockers (halothane, carbenoxolone, and octanol) in the presence or absence
of (GABAergic) synaptic transmission, it was demonstrated that populations of AME cells, inter alia
GABAergic interneurons, are coupled via gap junctions, enabling the cells to fire in synchrony with a
stable phase-difference. Therefore, gap junctions are a prerequisite for assembly formation by
synaptic interactions (Schneider and Stengl 2006). It remains to be investigated, whether other
mechanisms like neuron-glia-interactions also play a role for synchronization. Remarkably, in the
mammalian pacemaker center, the SCN, similar mechanisms are employed (Schneider and Stengl
2006): The neurons were also shown to fire very regularly and to be synchronized and phase-locked
in assemblies via gap junctions and GABA, which is the principal neurotransmitter of the SCN (Moore
and Speh 1993; Liu and Reppert 2000; Shinohara et al. 2000b; Schaap et al. 2003; Colwell 2005; Long
et al. 2005).
1.4.10.2
PDF synchronizes AME neurons
The effect of PDF on the electrical activity of AME-neurons was tested in extracellular recordings
(Schneider and Stengl 2005). PDF was shown to transiently activate or inhibit the spiking of the cells.
Because inhibitions but not activations could be reproduced in the absence of synaptic transmission,
inhibitions were suggested to be direct effects and activations to be indirect effects, i.e. inhibitions of
inhibitory neurons (disinhibitions). PDF effects in the presence of PTX indicated that (PTX-sensitive)
chloride channels were not involved in the PDF signal transduction cascade. Most interestingly, PDF
was shown to transiently synchronize different assemblies of AME-neurons to a new assembly. Cells,
which fired with a stable phase-difference before PDF application, were phase-locked PDFdependently. It was hypothesized, that PDF-dependent and in general peptide-dependent, transient
assembly formation is used in the AME to gate outputs via resonance, an important mechanism for
temporal encoding (Izhikevich et al. 2003; Schneider and Stengl 2005). According to this, PDFdependent disinhibition could phase-lock presynaptic cells, enabling them to spike at the same time
and thus to facilitate activation of postsynaptic cells, since the combined phase-locked input more
likely drives subthreshold membrane potential oscillations (SMPOs) of the postsynaptic cell to spike
threshold. In this way PDF could activate outputs to locomotor control centers. Thus, phase-control
on the timescale of milliseconds was suggested to be an essential mechanism of the circadian system
of the cockroach (Schneider and Stengl 2005).
1.4.10.3
AME neurons show a predominant period length of 2 hours
Since PDF-dependent assembly formation was accompanied by prominent changes in the mean
frequency of the recorded neurons (Schneider and Stengl 2005), it was suggested that fast, transient
frequency changes could indicate peptide release and peptide-dependent synchronization in the
AME (Schneider and Stengl 2007). Therefore, the daytime-dependent distribution of activity peaks in
the electrical multiunit activity, independent of the absolute mean frequency, was investigated in
recordings in DD. Maximum activity peaks occurred in the middle of the subjective night around ZT
18 while minimum activity peaks occurred in the middle of the subjective day around ZT 6, indicating
a circadian rhythm. Moreover, a predominant ultradian period length of 2 h and integer multiples of
40
Introduction
it (6 h) were found, causing further activity peak accumulations at the beginning, middle, and end of
the subjective day or the subjective night (Schneider and Stengl 2007). The AME-neurons were
suggested to generate rhythms with different period lengths, ranging from circadian rhythms to
ultradian rhythms in the timescale of hours and even milliseconds, possibly by different coupling
interactions between both AMAE, between pacemaker neurons of the AME, and between AMEneurons and downstream neurons (Klevecz et al. 1991; Schneider and Stengl 2005, 2006, 2007).
1.4.10.4
Ion channels involved in the generation of spontaneous activity
The generation of spontaneous activity was further investigated on the level of single AME cells (Petri
and Stengl 1999) in calcium imaging experiments (Wei and Stengl 2012). Since voltage-activated Ca2+
channels (VACCs) are activated when the cell depolarizes, calcium concentration changes may reflect
the electrical activity of the cell, with transient, high-amplitude changes indicating spikes and
sustained, low-amplitude changes indicating SMPOs (Moreaux and Laurent 2007, 2008; Wei and
Stengl 2012). The study of Wei and Stengl (2012) concentrated on the proportion of AME cells (26 %)
showing spontaneous, rapid calcium transients. The effects of different ion channel blockers in
combination with ion exchange experiments were integrated into a model for the interaction of
different ion channels in spontaneous activity generation (Fig. 15): Slowly inactivating, TTX-blockable
Na+ channels appear to play just a minor role as pacemaker channels. Instead, mibefradil-sensitive,
low voltage-activated (LVA) VACCs and DK-AH269-sensitive HCN channels were shown to be
pacemaker channels that depolarize the cells at negative resting potentials thereby driving the cells
to spike threshold. Spikes trigger nifedipine-sensitive high voltage-activated (HVA) L-type VACCs
which underlie the prominent calcium transients and are coupled to apamin-sensitive small
conductance-calcium-activated potassium channels (SKs), restraining the activity. Mibefradilsensitive LVA VACCs were suggested to be inhibited by Na+ influx during spike depolarization, thus
limiting Ca2+ influx. SKs and presumably other K+ channels are activated by the depolarization and/or
the increasing intracellular calcium concentration ([Ca2+]i) and repolarize the cells. Ion pumps were
suggested to return [Ca2+]i and [Na+]i eventually back to baseline levels. Additional ω-agatoxin IVAsensitive P/Q-type VACCs were found, which coupled to iberiotoxin-sensitive big conductancecalcium-activated potassium channels (BKs). However, these channels were suggested to be
exclusively involved in input-dependent activity (Wei and Stengl 2012).
Fig. 15. Proposed ion channels of AME neurons.
Calcium
imaging
experiments
suggested
the
involvement of certain ion channels in the regulation of
spontaneous activity and input-dependent activity. For
details see text. Abbreviations: BK/SK: large/small
2+
+
conductance-Ca -activated K channels, e/i: extracellular/intracellular side, HCN: hyperpolarizationactivated cyclic nucleotide-gated channels, HVA/LVA:
high/low voltage-activated, P/Q: P/Q-type, VACC:
2+
voltage-activated Ca channels, VASC: voltage-activated
+
Na channels. Redrawn and modified after (Wei and
Stengl 2012).
41
Introduction
1.5 Peripheral circadian clocks
Next to the central circadian clock, which controls systemic behavioral rhythmicity, circadian clocks
can also be found in a multitude of tissues, which control temporal processes in the respective tissue
(Reviews: Giebultowicz 2000; Glossop and Hardin 2002; Dibner et al. 2010; Tomioka et al. 2012).
These peripheral clocks probably occur in all organisms and have been studied particularly well in
mammals and insects.
1.5.1 Mammalian peripheral clocks
In mammals peripheral clocks can be found in skeletal muscles, brain parts outside the SCN, eye,
heart, kidney, liver, lung, spleen, pancreas, stomach, fibroblasts, pineal gland, thyroid gland, and
adrenal gland (Glossop and Hardin 2002; Dibner et al. 2010). In these clocks molecular oscillations
continue in vitro but dampen faster than in the SCN (Yamazaki et al. 2000). However, molecular
oscillations in isolated liver and lung tissue may also persist for more than 20 days (Yoo et al. 2004).
In vivo peripheral clocks continue cycling in SCN lesioned rodents, but synchrony between cells of
one tissue and between different tissues fades (Granados-Fuentes et al. 2004; Yoo et al. 2004).
Therefore, mammalian peripheral clocks contain self-sustained oscillators and require the SCNmaster-clock for entrainment, which may occur via direct or indirect cues from the SCN, such as
neuronal signals, hormones, body temperature, or food intake (Review: Dibner et al. 2010).
1.5.2 Peripheral clocks in insects
1.5.2.1 Peripheral clocks of D. melanogaster
In insects, several tissues and organs contain peripheral pacemakers. Already in 1988 per expression
was shown in antennae, proboscis, eyes, optic lobes, gut, malpighian tubules, and ovarian follicle
cells of D. melanogaster, suggesting that all these tissues may have intrinsic oscillator activity (Liu et
al. 1988). Indeed, in the following years daytime-dependent functions and molecular oscillations
were shown for several tissues and cell types: the larval ring gland complex, which contains the
prothoracic gland and controls eclosion (Emery et al. 1997; Myers et al. 2003), the malpighian
tubules controlling excretory activity (Liu et al. 1988; Giebultowicz and Hege 1997; Hege et al. 1997;
Plautz et al. 1997; Giebultowicz et al. 2000; Ivanchenko et al. 2001), epidermis cells controlling cuticle
deposition (Ito et al. 2008), photoreceptors controlling visual sensitivity (Siwicki et al. 1988; Zerr et al.
1990; Cheng and Hardin 1998; Barth et al. 2010), the testes and associated tissues controlling sperm
preparation (Plautz et al. 1997; Beaver et al. 2002), the oenocytes controlling synthesis and display of
pheromones (Krupp et al. 2008; Krupp et al. 2013), and chemosensory cells of the antennae,
proboscis, legs, and anterior wing margins controlling the sensitivity of these organs (Plautz et al.
1997; Tanoue et al. 2004; Chatterjee et al. 2010, 1.5.3.7). Most of these tissues were shown to
continue molecular cycling in vitro under constant conditions or in vivo without functional central
42
Introduction
pacemaker neurons and to be entrainable to new light or temperature cycles (Glaser and Stanewsky
2005), thus demonstrating autonomous oscillator function. As shown for mammals (Yamazaki et al.
2000), molecular oscillations of isolated peripheral clocks dampen faster than those of central
pacemaker neurons (Hardin 1994; Plautz et al. 1997), indicating a requirement for a certain degree of
input from the latter. This input may be provided by neuropeptides such as PDF, as shown for the
prothoracic gland clock, where PDF release from the central LNvs is required for synchronized
molecular oscillations (Myers et al. 2003). In contrast, in the oenocyte clock PDF is not required for
robust cycling, but PDF released from the central LNvs as well as the PDF-ir cells of the abdominal
ganglia (see 1.3.5) transmits phase-information to the oenocyte clock and regulates its period and its
physiological output, the expression of cuticular hydrocarbons, which function as sex pheromones
(Krupp et al. 2008; Krupp et al. 2013). Since loss of PDF also affects the mating activity in a sexspecific manner, it was suggested that PDF may be required to couple pheromone synthesis and
mating (Krupp et al. 2013). However, other peripheral clocks, like chemosensory cells of the
antennae or the proboscis (Tanoue et al. 2004; Chatterjee et al. 2010), appear to function
autonomously without central modulation. In general, in D. melanogaster and other cry1 expressing
insects the need for entrainment or synchronization via the central pacemaker is not as high as in
mammals, since light may penetrate the cuticle and allow for CRY1-dependent entrainment of the
respective peripheral clocks in a cell autonomous manner (Glossop and Hardin 2002) or indirectly via
unknown pathways as shown for the epidermis clock (Ito et al. 2008).
1.5.2.2 Differences between the central and peripheral molecular clocks
Remarkably, the molecular mechanisms used to generate circadian oscillations in the fruit fly’s
peripheral clocks may differ from those of the central clock, with the most obvious difference being
that CRY1 does not only function as blue light photoreceptor but also as a core clock component.
However, this does not apply to the epidermis clock (Ito et al. 2008; Tomioka et al. 2012). Already in
the description of the cryb mutation it was noticed that this mutation affected central and peripheral
oscillators in a different way (Stanewsky et al. 1998). In later studies performed on malpighian
tubules (Ivanchenko et al. 2001), antennae (Krishnan et al. 2001; Levine et al. 2002b), and larval
prothoracic glands (Myers et al. 2003) phenotypes such as loss of rhythmicity in DD were observed
that were not explainable by a lack of photoreception, indicating a core clock function for CRY1.
Indeed, a role for CRY1 in endogenous phase regulation and thus regulation of gene expression was
suggested for the antennal clock (Levine et al. 2002b) and a PER-dependent function as
transcriptional repressor of CLK/CYC-mediated gene expression was demonstrated in photoreceptors
(Collins et al. 2006).
1.5.2.3 Peripheral clocks in other insects than D. melanogaster
Peripheral clocks were also demonstrated for other insects than D. melanogaster. For the cockroach
R. maderae for example peripheral clocks were suggested to control cuticle deposition (Wiedenmann
et al. 1986) and the sensitivity of olfactory receptor neurons (ORNs) of the antenna (Saifullah and
Page 2009, 1.5.3.7) and for the cricket Gryllus bimaculatus peripheral clocks were shown in brain,
43
Introduction
terminal abdominal ganglion (TAG), midgut, and anterior stomach (Uryu and Tomioka 2010). These
insects do not appear to express cry1 and thus, do not appear to possess cell autonomous light
entrainment, but entrain solely via opsin-based photoreceptors of the complex eyes. Therefore,
peripheral clocks of these species need to be entrained via the central pacemaker or via other
unknown pathways from the complex eyes (Tomioka et al. 2012). In addition to cockroaches and
crickets many studies concentrated on lepidopteran species: These studies mainly focused on the
antennal clock, but also clocks in photoreceptors (Sauman and Reppert 1996) or cells of the male
reproductive system were described (Giebultowicz et al. 1989; Gvakharia et al. 2000).
1.5.3 The antennal clock of insects
Given that the antennal clock is the best studied peripheral clock of insects so far and that a major
part of the experimental work of this thesis deals with the antennal clock, this chapter shall provide a
short overview of the present knowledge on antennal clocks of different insect species. In addition,
the function of the antennae, which is temporally regulated by these clocks, is described.
1.5.3.1 Antennae, the insects' nose
Sensitive detection of volatile molecules in an insect’s environment is of great importance, since
these molecules (odorants) might provide information about food sources, predators, or conspecifics
and potential mating partners in the case of intraspecific signaling molecules (pheromones).
Therefore, insects have evolved a highly sensitive sense of smell (olfaction), as it becomes obvious
when tens of fruit flies accumulate on a ripe fruit, when mosquitoes are highly attracted to human
skin odor, or when a male moth exhibits its typical upwind zig-zagging and casting behavior,
following filaments of the species-specific pheromone blend, released by a ready-to-mate female
over a long distance.
Insects detect odorants with their antennae and maxillary palps, on whose surface hundreds to
thousands of sensilla can be found, such as different types of olfactory sensilla (sensilla trichodea,
basiconica, placodea, or coeloconica) as well as sensilla of other sensory modalities (Fig. 16, von
Frisch 1921; Schneider 1969; Lee and Strausfeld 1990; Stocker 1994; Hallberg and Hansson 1999;
Shanbhag et al. 1999). Each sensillum houses a certain number of ORNs, which ranges from one to
four in D. melanogaster (Stocker 1994; Shanbhag et al. 1999) and may amount to up to 30 in other
insects (Review: Nakagawa and Vosshall 2009). Pheromone-sensitive trichoid sensilla of M. sexta
house two ORNs (Keil 1989), one of which responds to bombykal (BAL = (E,Z)-10,12-hexadecadienal),
the main component of the sex pheromone blend. The second ORN responds to one of the minor
components such as (E,E,Z)-10,12,13-hexadecatrienal (Starratt et al. 1979; Kaissling et al. 1989;
Tumlinson et al. 1989; Kalinova et al. 2001). Next to the ORNs three types of supporting cells can be
found: The thecogen cell isolates soma and inner dendrite of the ORN, thereby ensuring that only the
outer dendrite gets into contact with the sensillum lymph. The trichogen and the tormogen cells
contribute to the unique composition of the sensillum lymph (Fig. 17, Keil 1989).
44
Introduction
Fig. 16. Antennae of the hawkmoth M. sexta. A. Male M. sexta with laid-back antennae (arrow) resting on a leaf. Photo
taken by Thomas Hörbrandt, University of Regensburg. B. Lateral view of the antenna, modified after (Keil 1989). The rear
side (= leeward) of the antenna is covered with large scales (asterisk). C. Two annuli of the antenna, photographed from
the frontal side (= windward), modified after (Lee and Strausfeld 1990). The long sensilla trichodea are arranged in basketlike arrays (arrowheads in B and C), optimizing the capture of pheromone molecules. Scale bar: 100 µm
In flying insects like moths, with a wing beat frequency of 20 - 30 Hz, each downstroke of the wings
accelerates the air flow over the antennae and allows for periodic odor sampling comparable with
the mammalian "sniffing" (Loudon and Koehl 2000; Sane 2006; Sane and Jacobson 2006; Tripathy et
al. 2010; Daly et al. 2013; Review: Stengl 2010). In walking insects flicking of the antennae might
serve a similar purpose (Stengl and Funk 2013). Before odorants can get into contact with their
receptors, certain "perireceptor events" take place (Reviews: Kaissling 2009, 2013): Odorants are
adsorbed on the waxy cuticle of the sensillar arrays and may enter the interior of the sensilla via
pores in the cuticula (Fig. 17). Odorant or pheromone binding proteins (OBPs, PBPs), which are
secreted in millimolar concentrations by supporting cells of the sensillum (Fig. 17), bind and
transport the lipophilic odorants or pheromones through the aqueous sensillum lymph towards the
ORNs. Alternatively these proteins, together with odorant degrading enzymes (ODEs), might be
implicated in the degradation of odorants (Reviews: Stengl et al. 1992a; Stengl 2010). At least
pheromones appear to interact with additional molecules, the sensory neuron membrane proteins
(SNMPs), whose function is not fully clarified (Rogers et al. 1997; Rogers et al. 2001a; Rogers et al.
2001b; Benton et al. 2007; Forstner et al. 2008; Liu et al. 2014a; Review: Vogt et al. 2009). SNMPs are
related to the vertebrate CD36 protein, a scavenger receptor, involved in lipoprotein binding and
uptake of fatty acids, carotenoids, and cholesterol esters (Bonen et al. 2004; Ge and Elghetany 2005),
and are assumed to play a role in pheromone recognition, since SNMP-1 expression was shown to be
required for the response to the aggregation pheromone 11-cis-vaccenyl acetate (cVA) in
D. melanogaster in vivo (Benton et al. 2007; Jin et al. 2008, 1.5.3.8) and coexpression of H. virescens
SNMP-1 in a heterologous expression system dramatically increased the sensitivity of pheromone
responses (Pregitzer et al. 2014). In the moths H. virescens and A. polyphemus SNMP-1 is expressed
in pheromone-sensitive ORNs, while SNMP-2 is expressed in supporting cells, indicating different
functions. However, in D. melanogaster SNMP-1 is expressed in pheromone-sensitive ORNs and
additionally in supporting cells throughout the antenna (Benton et al. 2007) and in different moths
both SNMP-types are abundant in the antennae of both sexes and are also expressed in cells of
basiconic sensilla, suggesting an involvement in general odorant detection (Rogers et al. 2001a; Liu et
al. 2014a).
45
Introduction
Fig. 17. Pheromone-sensitive trichoid sensillum of
M. sexta. Each sensillum houses two olfactory receptor
neurons (ORNs), which extend their outer dendrites into
the sensillum lymph (SL). Pheromones may enter the
sensillum via pores (P) in the cuticle (CU) containing pore
tubules. Next to the ORNs three types of supporting cells
are present: The thecogen cell (TE) isolates the ORNs' inner
dendrites and somata from the sensillum lymph, while the
tormogen cell (TO) and trichogen cell (TR) contribute to the
composition of the sensillum lymph, which is different from
the composition of the hemolymph (HL) beyond the basal
lamina. The ORNs' axons, which project into the brain's
antennal lobe, are isolated by glial cells (GL). Figure and
legend modified after (Stengl and Funk 2013).
1.5.3.2 Odorants are sensed with different types of receptors
The molecular basis of odorant detection is best studied in D. melanogaster, where ORNs express
different types of chemosensory receptors (Getahun et al. 2012; Review: Silbering and Benton 2010).
Ionotropic receptors (IRs) represent an evolutionary ancient class of receptors, which have evolved
from ionotropic glutamate receptor (IGluR) ancestors and seem to be present across the protostomia
clade of the animal kingdom (Benton et al. 2009; Croset et al. 2010; Review: Rytz et al. 2013). IRs are
expressed in ORNs of coeloconic sensilla and are tuned to the detection of acids, ammonia, and
humidity. They form heteromeric complexes, suggested to allow for ionotropic olfactory transduction
(Benton et al. 2009; Abuin et al. 2011; Ai et al. 2013). Gustatory receptors (GRs) are mainly expressed
in gustatory organs like the proboscis, but two GRs required for CO2 detection (GR21a and GR63a)
are expressed in an antennal ORN (ab1c, Kwon et al. 2007). The third group are the classical insect
odorant receptors (ORs), which are tuned to odorants of various chemical classes (Clyne et al. 1999;
Vosshall et al. 1999; Vosshall et al. 2000; Hallem et al. 2004a; Couto et al. 2005). In D. melanogaster
ORs are expressed in ORNs innervating basiconic (ab) and trichoid sensilla (at) of the antennae as
well as basiconic sensilla of the maxillary palps (pb, Couto et al. 2005), with the exception of OR35a,
which is expressed together with IRs in coeloconic sensilla (Yao et al. 2005; Abuin et al. 2011). This
class of receptors evolved later than IRs and GRs and appears to be specific for insects (Croset et al.
2010). Consistently, no proteins of the insect OR family were detected so far in crustaceans (PeñalvaArana et al. 2009; Corey et al. 2013; Groh et al. 2013). Insect ORs are a divergent family of seventransmembrane domain (7TM) proteins, with sequence identities amongst each other as low as 17 %
for some insect species (Clyne et al. 1999; Vosshall et al. 1999; Hill et al. 2002; Krieger et al. 2002;
Vosshall 2003). Surprisingly, the proteins of this family are different from all other known GPCRs and
have no amino acid homology with ORs from Caenorhabdidits elegans or vertebrates (Wistrand et al.
2006; Reviews: Nakagawa and Vosshall 2009; Stengl and Funk 2013). Computational as well as
experimental evidence suggested that insect ORs have adopted an inverted membrane topology
compared to other GPCRs, with an intracellular N-terminus and an extracellular C-terminus (Benton
46
Introduction
et al. 2006; Wistrand et al. 2006; Lundin et al. 2007; Smart et al. 2008; Guo and Kim 2010; Tsitoura et
al. 2010; Zhou et al. 2013), raising the question of whether these proteins possess an unknown
G protein binding site, or whether G protein-independent signaling pathways are employed
(see 1.5.3.6). Interestingly, the same atypical topology was shown for the related family of insect GRs
(Zhang et al. 2011).
1.5.3.3 The coreceptor ORCO is required for membrane insertion of
conventional ORs
In contrast to vertebrates, where each ORN expresses only one OR, apparently due to negative
feedback regulation (Serizawa et al. 2003; Lewcock and Reed 2004), in D. melanogaster more than
one functional OR might be expressed per ORN, as shown for OR33c and OR85e, which are
coexpressed in pb2a neurons (Goldman et al. 2005). Additionally, an ubiquitous coreceptor is
coexpressed along with conventional ORs. It was first identified in D. melanogaster and named
OR83b (encoded by the gene a45, Vosshall et al. 1999; Vosshall et al. 2000). Due to the extraordinary
high amino acid-sequence identity of up to 100 % for closely related species (Hull et al. 2012) OR83borthologues were also identified in other insect species and given names like OR7 in mosquitoes (Hill
et al. 2002) or OR2 in moths (Tab. 5, Krieger et al. 2002; Krieger et al. 2003; Patch et al. 2009). In
order to simplify matters a unified nomenclature was concluded and the gene was renamed olfactory
receptor coreceptor (orco, Vosshall and Hansson 2011). ORCO appears to be expressed only in ORNs
that express ORs, but neither in ORNs that solely express IRs or GRs nor in sensory neurons of
mechanosensory sensilla (Larsson et al. 2004). In other insect species ORCO expression is also
restricted to olfactory sensilla (Krieger et al. 2003; Pitts et al. 2004; Jones et al. 2005; Nakagawa et al.
2005). Additionally, in moths and mosquitoes orco expression was detected in gustatory organs like
legs and proboscis (Krieger et al. 2002; Melo et al. 2004; Pitts et al. 2004; Xia and Zwiebel 2006).
However, an additional gustatory function of ORCO seems unlikely, since these gustatory organs also
express ORs, as shown for the proboscis of Anopheles gambiae (Kwon et al. 2006) and H. virescens
(Krieger et al. 2002), suggesting a close-range olfactory function of these tissues. Previously,
functional ORCO expression was detected in the flagellum of spermatozoa of different insect species,
where it is most probably coexpressed with ORs, playing a role in the detection of chemical signals
(Pitts et al. 2014).
Analysis of orco null mutants or flies, in which orco expression was knocked down using RNAi,
revealed severe olfactory deficits on the behavioral and physiological level (Larsson et al. 2004;
Neuhaus et al. 2005). In these flies the localization of ORs to the outer dendritic ORN-membrane was
severely disrupted. Given the undisputable requirement for the receptors at the site, where signal
transduction takes place, the observed olfactory deficits are a logical consequence of the loss of orco.
Several studies could demonstrate physical interaction between ORCO and ORs: Homo- as well as
heteromers of unknown stoichiometry were detected in vitro and in vivo (Neuhaus et al. 2005;
Benton et al. 2006; Tsitoura et al. 2010; German et al. 2013). The complexes were suggested to be
associated via the conserved C-terminal domains TM4 - TM7, most probably via interactions between
their cytoplasmic IC3-loops (Benton et al. 2006; Miller and Tu 2008; Harini and Sowdhamini 2012). In
vivo, formation of heteromeric complexes was shown to occur early in the endomembrane system
(prior to arrival at the Golgi apparatus) and to be required for the ciliary trafficking pathway.
47
Introduction
Moreover, ORCO was not only shown to be required for dendritic OR localization, but also for stable
maintenance at this location (Benton et al. 2006).
Tab. 5. Orco orthologues of different insect species, modified after (Stengl and Funk 2013).
Species
Acromyrmex echinatior
Acyrthosiphon pisum
Aedes aegypti
Aldrichina grahami
Amyelois transitella
Anopheles gambiae
Antheraea pernyi
Apis cerana cerana
Apis mellifera
Apocrypta bakeri
Apolygus lucorum
Bactrocera cucurbitae
Bactrocera dorsalis
Bombyx mori
Calliphora erythrocephala
Ceratitis capitata
Ceratosolen solmsi marchali
Chrysomya megacephala
Chrysomya rufifacies
Culex quinquefasciatus
Diaphania indica
Drosophila ananassae
Drosophila pseudoobscura
Drosophila yakuba
Epiphyas postvittana
Haematobia irritans irritans
Harpegnathos saltator
Helicoverpa armigera
Helicoverpa assulta
Helicoverpa zea
Heliothis viriplaca
Heliothis virescens
Holotrichia oblita
Holotrichia plumbea
Locusta migratoria
Lygus hesperus
Lygus lineolaris
Lucilia sericata
Manduca sexta
Microplitis mediator
Musca domestica
Mythimna separata
Nasonia vitripennis
Ostrinia nubilalis
Ostrinia scapulalis
Pediculus humanus corporis
Philotrypesis pilosa
Philotrypesis sp. BL-2007
Plutella xylostella
Schistocerca gregaria
Sitobion avenae
Spodoptera frugiperda
Spodoptera littoralis
Spodoptera litura
Stomoxys calcitrans
Tenebrio molitor
Tribolium castaneum
Original
denotation
OR7
ApOr1
AaOr7
AgraOrco
AtraOrco
AgamGPRor7
AperR2
AcerOR2
AmelR2
AbOr2
AlucOrco
Or83b
BdOrco
BmorR2
CeryR2
CcOr83b
CsmOr2
CmegOrco
CrufOrco
CqOR7
DiOR83
DanaOrco
Or83b
Dpse\Orco
DyakOrco
EpOR2
HirrOrco
Hsal\Orco
OR83b
HassOrco
HzOr83b
HvirOr83b
HR2
HoblOrco
Or83b
LmigOrco
LhOrco
LlOrco
LserOR1
MsextaOR2
MmedOrco
MdomOrco
MsOR83
NvOr1
OnOr2
OscaOR2
PhumOrco
PpOr2
PsOr2
PxOR83
SgreOrco
SaveOrco
SfOR2
Slit-R2
SlitOrco
ScalOrco
TmolR2
TcOR1
Accession
number
EGI63650
XP_001951646
AY582943
HQ190955
JX173647
AY363725
AJ555486
JN792581
NM_001134943
EU281849
KC881255
HM745934
EU621792
AJ555487
AJ555538
AY843206
EU281848
HQ315861
JQ365176
DQ231246
AB263114
XM_001953308
AY567998
XM_001359327
XM_002096017
EU791887
ACF21678
EFN84180
HQ186284
EU057178
AY843204
AFI25169
AJ487477
JF718662
HQ110087
JN989549
JQ639213
JQ639214
HQ315862
FJ546087
EF141511
JQ365179
AB263111
NM_001170994
GQ844877
AB467318
EEB12924
EU281850
EU281851
AB263117
JN989550
GQ275379
EF395366
DQ845292
EU622914
AJ555539
AM689918
48
Reference
(Hull et al. 2012)
(Smadja et al. 2009)
(Melo et al. 2004)
(Olafson 2013)
(Xu et al. 2012)
(Hill et al. 2002)
(Krieger et al. 2003)
(Zhao et al. 2013)
(Lu et al. 2009)
(Zhou et al. 2013)
(Zheng et al. 2012)
(Zheng et al. 2012)
(Jones et al. 2005)
(Lu et al. 2009)
(Olafson 2013)
(Zhou et al. 2013)
(Xia and Zwiebel 2006)
(Mitsuno et al. 2008)
(Olafson 2013)
(Vosshall et al. 2000)
(Zhou et al. 2013)
(Olafson 2013)
(Jordan et al. 2009)
(Olafson 2013)
(Jones et al. 2011)
(Zheng et al. 2012)
(Yang et al. 2012)
(Jones et al. 2005)
(Ma et al. 2013)
(Yang et al. 2012)
(Zheng et al. 2012)
(Yang et al. 2012)
(Hull et al. 2012)
(Hull et al. 2012)
(Wang et al. 2012)
(Patch et al. 2009)
(Li et al. 2012)
(Olafson 2013)
(Robertson et al. 2010)
(Wanner et al. 2010)
(Miura et al. 2010)
(Yang et al. 2012)
(Lu et al. 2009)
(Lu et al. 2009)
(Yang et al. 2012)
(Yang et al. 2012)
(Smart et al. 2008)
(Merlin et al. 2007)
(Wu et al. 2013)
(Olafson 2013)
(Abdel-Latief 2007)
Introduction
1.5.3.4 OR/ORCO complexes function as ion channels in vitro
Different studies agreed that ORCO does not bind odorant-ligands and that the specificity of the
OR/ORCO complexes solely depend on the OR subunit (Dobritsa et al. 2003; Elmore et al. 2003;
Hallem et al. 2004a; Hallem et al. 2004b; Neuhaus et al. 2005; Sato et al. 2008; Wicher et al. 2008;
Jones et al. 2011; Pask et al. 2011; Chen and Luetje 2012). When ORs of different insect species were
heterologously expressed without ORCO, stimulation with odorants, mostly in very high
concentrations, provoked ligand-specific responses (Wetzel et al. 2001; Sakurai et al. 2004;
Nakagawa et al. 2005; Neuhaus et al. 2005; Grosse-Wilde et al. 2006; Kiely et al. 2007; Smart et al.
2008; Deng et al. 2011). Apparently, the ORs could form complexes with endogenously expressed
ORCO, which might be the case in S2 cells or Spodoptera frugiperda 9 (SF9) cells (Kiely et al. 2007;
Kiely 2008; Smart et al. 2008), or the ORs could couple to endogenous or coexpressed G proteins,
which might be the case in non-insect cells like HEK 293 cells or Xenopus laevis oocytes (Wetzel et al.
2001; Sakurai et al. 2004; Nakagawa et al. 2005; Neuhaus et al. 2005; Grosse-Wilde et al. 2006; Smart
et al. 2008; Deng et al. 2011). Previously, one study reported coupling of D. melanogaster OR22a
(DmelOR22a) to HEK 293 cell-endogenous Gαo and Gαi subunits (Ignatious Raja et al. 2014). However,
coexpression of an ORCO orthologue, not necessarily from the same insect species, increased the
number of responding cells and the sensitivity (Nakagawa et al. 2005; Neuhaus et al. 2005; Smart et
al. 2008). Remarkably, when ORCO instead of a G protein was coexpressed, the biophysical
properties of the odorant-evoked responses changed, suggesting an involvement of ORCO in signal
transduction (Nakagawa et al. 2005; Review: Nakagawa and Vosshall 2009). Indeed, following studies
clearly demonstrated, that OR/ORCO complexes may function as odorant-gated ion channels in vitro
(Sato et al. 2008; Smart et al. 2008; Wicher et al. 2008). The details and implications of this
mechanism are still controversially discussed (Review: Stengl and Funk 2013). When different ORs
and ORCO were coexpressed in heterologous systems, stimulation with odorants in high
concentration elicited non-selective cation currents, which apparently were independent of
G protein signaling and were suggested to result from odorant-induced ionotropic signal
transduction (Fig. 18, Sato et al. 2008; Smart et al. 2008; Wicher et al. 2008). Such an ionotropic
mechanism implies that odorant binding to the OR/ORCO complex leads to conformational changes
of the complex, activating an ion conductance of the complex, which is independent of G protein and
second messenger signaling. Since stimulation with different second messenger analogues did not
induce responses and interference with G protein signaling did not completely abolish odorantdependent responses, it was concluded, that insects solely employ ionotropic signal transduction to
detect odorants (Sato et al. 2008; Smart et al. 2008). The corresponding ion channel was suggested
to be formed by OR and ORCO subunits with unknown stoichiometry (Sato et al. 2008). However,
Wicher et al. provided evidence for two parallel signaling pathways: Odorant stimulation induced an
ionotropic current (Ii) and a metabotropic current (Im). The ionotropic current developed and
decayed faster than the metabotropic current and was dependent on approximately 100 times
higher odorant concentrations than the more sensitive metabotropic current, which was dependent
on the presence of ATP and GTP. Moreover, ligand binding to the conventional OR (OR22a) was
shown to increase the intracellular cAMP level, and the metabotropic current was shown to be
dependent only on ORCO but not on OR expression and to be sensitive to cAMP and cGMP (Wicher
et al. 2008). Thus, it was concluded that odorant binding to ORs might induce two signaling
pathways, one direct, ionotropic pathway and one slower but more sensitive, metabotropic pathway,
which involved coupling of the OR to Gαs, and cAMP-dependent activation of an ion channel solely
49
Introduction
formed by one or more ORCO subunits (Wicher et al. 2008). Additionally, phosphorylation of
D. melanogaster ORCO (DmelORCO) by protein kinase C (PKC) was shown to be required for its
cAMP-sensitivity and sufficient for its activation in the absence of cyclic nucleotides (Fig. 18, Sargsyan
et al. 2011; Getahun et al. 2013). Since PKC activation is dependent on PLCβ activity and thus on the
intracellular Ca2+ concentration, ORCO activity is also dependent on the intracellular Ca2+
concentration, providing an explanation for the lack of second messenger-dependent ORCO
activation in other studies (Sato et al. 2008; Jones et al. 2011).
Fig. 18. Current models for olfactory transduction. A. Odorants are bound by odorant binding proteins (OBPs) and
transported through the aqueous sensillum lymph. In the models of Sato et al. (2008) and Nakagawa and Vosshall (2009)
odorant binding to the odorant receptor (OR) induces exclusively an ionotropic transduction process, in which the
complete OR/ORCO complex functions as ion channel conducting an ionotropic transduction current (Ii). The OR/ORCO
2+
complex might be modulated by second messengers, protein kinases or Ca /calmodulin (CaM), which are initiated via
unknown pathways, independently from odorant binding and transduction. B. In the model proposed by Wicher et al.
(2008) odorant binding to the OR induces also a fast ionotropic transduction current (Ii), which is conducted through ORCO
alone. Simultaneously, odorant binding activates a trimeric Gs protein, leading to elevated cAMP levels, activating a slower,
more sensitive, metabotropic transduction current (Im). In addition, the protein kinase C (PKC) pathway is activated by
odorant stimulation (Getahun 2013), and phosphorylation of ORCO by PKC is a prerequisite for its cAMP sensitivity
(Sargsyan et al. 2011). Finally, ORCO-phosphorylation by CaM also contributes to sensitization (Mukunda et al. 2014).
Abbreviations: AC: adenylyl cyclase, ATP: adenosine trisphosphate, cAMP/cGMP: cyclic adenosine/guanosine
monophosphate, DAG: diacylglycerol, Gαs: stimulatory α-subunit, βγ: βγ-subunit of the trimeric G protein, IP3: inositol 1,4,5trisphosphate, PLCβ: phospholipase Cβ, PIP2: phosphatidylinositol 4,5-bisphosphate, PKA/PKC/PKG: protein kinase A/C/G.
Figure and legend modified after Stengl and Funk (2013).
50
Introduction
In the last years extensive research was performed, but the detailed mechanism of the OR/ORCO
complex is still not resolved. Different studies provided evidence that both, ORs and ORCO,
contribute to the biophysical properties of the complex and that the subunit composition affects the
sensitivity to the non-specific cation channel blocker ruthenium red, with the OR subunit being the
major determinant of sensitivity (Sato et al. 2008; Nichols et al. 2011; Pask et al. 2011; Nakagawa et
al. 2012). Some conserved amino acids were detected in DmelORCO (Wicher et al. 2008; Kumar et al.
2013) and in B. mori ORCO and OR1 (BmorORCO, BmorOR1, Nakagawa et al. 2012), whose
replacement affected the biophysical properties of the channel complex. However, it was not clearly
demonstrated whether ligand-binding ORs directly contribute to the pore of the ion channel complex
or whether they affected the pore only indirectly. Different non-specific antagonists such as
ruthenium red, lanthanum, 2-ABP, DEET, IR3535, and different amiloride derivatives (Nakagawa et al.
2005; Sato et al. 2008; Smart et al. 2008; Jones et al. 2011; Nichols et al. 2011; Pask et al. 2011;
Bohbot and Dickens 2012; Pask et al. 2013; Röllecke et al. 2013) and non-specific agonists such as
cyclic nucleotides, GTPγS, or activators of PLC or PKC were reported (Wicher et al. 2008; Deng et al.
2011; Nakagawa and Touhara 2013). Furthermore, ORCO specific agonists and antagonists were
identified, with the agonist VUAA1 (N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3yl)thio)acetamide) being the first one reported (Jones et al. 2011). VUAA1 was identified in a screen
of almost 120,000 compounds (Jones et al. 2011) and its agonistic function was confirmed for ORCO
orthologues of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, D. melanogaster,
Harpegnathos saltator, Heliothis virescens, and Ostrinia nubilalis (Jones et al. 2011; Pask et al. 2011;
Bohbot and Dickens 2012; Chen and Luetje 2012; Jones et al. 2012; Taylor et al. 2012; Kumar et al.
2013). Structural modification of VUAA1 (Fig. 30) revealed further agonists, some of them being even
more potent than VUAA1, and also produced antagonists (Bohbot and Dickens 2012; Chen and Luetje
2012; Jones et al. 2012; Taylor et al. 2012; Chen and Luetje 2013). Unfortunately, all these
compounds' chemical properties do not allow for volatile delivery, thereby drastically limiting the
field of application (Chen and Luetje 2013). Given the chemical similarity, probably all ORCO agonists
might synergistically interact with odorants (i.e. the effects of odorant and ORCO agonist add up and
might promote each other), as shown for ORCO receptor activator molecule 2 (OrcoRAM2, Bohbot
and Dickens 2012). The common mechanism of action of specific ORCO antagonists might be a
competitive inhibition of ORCO activation and a non-competitive inhibition of odorant-induced OR
activation (Chen and Luetje 2012; Jones et al. 2012; Chen and Luetje 2013).
1.5.3.5 ORCO as well as conventional ORs regulate spontaneous activity
Although the functions of ORCO are a controversial issue, several studies agreed on the influence of
ORCO on spontaneous activity: In D. melanogaster each type of ORN exhibits a distinct pattern of
spontaneous activity in vivo, which might be increased or decreased upon odorant stimulation (de
Bruyne et al. 1999; de Bruyne et al. 2001; Hallem et al. 2004a). When ORs and ORCO were
coexpressed in heterologous systems, the resulting complexes mediated spontaneous Ca2+ influx
(Sato et al. 2008; Wicher et al. 2008) and even in the absence of ORs spontaneous openings of
channels formed by ORCO were detected (Jones et al. 2011), providing a higher resting current
compared with untransfected control cells (Sargsyan et al. 2011). Consistently, ORNs of orco mutant
flies were shown to exhibit barely spontaneous activity (Larsson et al. 2004; Benton et al. 2007; Deng
et al. 2011), suggesting that spontaneously active ORCO provides a leak current, which is required to
51
Introduction
depolarize hyperpolarized ORNs to spike threshold, thereby facilitating spontaneous spiking (Stengl
2010; Stengl and Funk 2013). ORCO-dependent regulation of spontaneous activity was also
demonstrated by specific ORCO-agonists, which elevated spontaneous activity in ORNs of A. gambiae
(Jones et al. 2011) and D. melanogaster (Su et al. 2012), or ORCO-antagonist, which decreased
spontaneous activity in ORNs of A. gambiae (Jones et al. 2012). However, conventional ORs also
contribute to spontaneous activity, since loss or replacement of an OR in vivo changed the specific
pattern of spontaneous activity of the ORN (Dobritsa et al. 2003; Elmore et al. 2003; Hallem et al.
2004a). Consistently, the current recorded under control conditions in HEK 293 cells expressing
OR22a and ORCO was decreased by an OR22a inhibitor (Wicher et al. 2008). Thus, the strong
reduction of spontaneous activity in orco mutant flies might not only be due to the lack of ORCO but
additionally to the impaired localization of ORs to the dendritic membrane. Remarkably, not only the
frequency and the pattern of spontaneous activity are OR-dependent, but also the spike amplitude
(see 1.5.3.7, Krishnan et al. 2008).
Due to the limited number of studies performed in vivo it remains difficult to estimate the
significance of the data, which were collected mainly in heterologous expression systems. One critical
point clearly is the slow time course of the odorant-induced responses in vitro, which peaked and
terminated in the range of seconds (Ii and Im, Wicher et al. 2008; Sargsyan et al. 2011), while odorant
responses in vivo take place in the range of milliseconds. The shortest latency, reported for odorantinduced ionotropic signaling, was around 20 ms (Sato et al. 2008) and thus matched kinetics of
metabotropic signaling cascades, whose components are linked by signalosomes, as reported for
visual signal transduction in D. melanogaster (Hardie and Raghu 2001), but not the kinetics of typical
ionotropic signaling, which take places in the range of microseconds (Reviews: Stengl 2010; Stengl
and Funk 2013). In one in vivo study, in which genetic interference with different Gα subunits was
investigated, no severe effects on odorant responses were detected with the employed data analysis,
which did not support a role for G proteins in olfactory transduction (Yao and Carlson 2010).
However, only one Gα subunit and thus only one metabotropic signaling cascade at a time was
affected and most probably parallel signaling cascades might substitute for the impairment (Stengl
and Funk 2013). In contrast, the importance of PKC-dependent ORCO phosphorylation and its cyclic
nucleotide sensitivity was also demonstrated in vivo (Olsson et al. 2011; Sargsyan et al. 2011;
Getahun et al. 2013). The results clearly indicated an ORCO-dependent sensitization mechanism of
general odorant responses. It was suggested that metabotropic tuning mechanisms are employed to
expand the response range of ionotropic signal transduction in D. melanogaster (Getahun et al.
2013).
1.5.3.6 Odorant-induced metabotropic signaling cascades
Several studies provided evidence for the involvement of various G protein dependent signaling
cascades in insect olfaction. Different G proteins and components of the associated signaling
pathways were shown to be expressed in insect antennae or even in the ORNs themselves.
Overexpression, stimulation, or impairment of these signaling components often severely affected
odorant responses, suggesting several parallel signaling cascades. These might be employed
differentially due to the physiological state of the insect and the nature of the odorant stimulus
(Tab. 6, Review: Stengl 2010). Clearly, there is metabotropic modulation of odorant responses, as
52
Introduction
shown for the sensitizing or adapting effects of cyclic nucleotides. Since the concentration of cGMP
rises after strong, adapting pheromone stimuli (Ziegelberger et al. 1990; Boekhoff et al. 1993; Stengl
et al. 2001), it was suggested to be involved in adaptation during the resting phase of M. sexta
(Flecke et al. 2006). The stress hormone octopamine, whose hemolymph levels oscillate in M. sexta
(Lehman 1990) as well as Trichoplusia ni (Linn Jr et al. 1994b; Linn Jr et al. 1994a; Linn et al. 1996),
was suggested to mediate sensitization, at least partly in a cAMP-dependent manner (Flecke and
Stengl 2009; Flecke et al. 2010). Therefore, it was suggested, that cAMP and cGMP levels in the
antennae display daytime-dependent changes, involved in the control of olfactory sensitivity
(see 1.5.3.7, Flecke et al. 2006; Flecke and Stengl 2009; Flecke et al. 2010; Stengl 2010). Consistently,
daytime-dependent changes in octopamine, cAMP, and IP3 levels were detected in M. sexta
antennae, and stimulation with octopamine elevated the concentrations of cAMP and IP3
(Schendzielorz et al. 2015). In contrast, other studies directly implicated G protein-dependent
signaling pathways in olfactory transduction. As mentioned above, insect ORs were shown to directly
or indirectly couple to G proteins when expressed in heterologous expression systems (Wetzel et al.
2001; Sakurai et al. 2004; Nakagawa et al. 2005; Neuhaus et al. 2005; Grosse-Wilde et al. 2006; Kiely
et al. 2007; Smart et al. 2008; Deng et al. 2011), particularly when a Gα subunit is coexpressed, which
ensures coupling to the cell endogenous signaling machinery, such as the Gα15 or Gα16 subunits
(Offermanns and Simon 1995). Deng et al. (2011) generated different chimeric G proteins composed
of the human Gα16 N-terminus and the C-terminus of different D. melanogaster Gα subunits. When
the chimeric G proteins were coexpressed with DmelOR22a in HEK 293 cells, odorant responses were
improved for each Gα subunit, with the Gαs-chimera providing the strongest improvement.
Apparently, the fruit fly-specific Gαs N-terminus improved coupling to the OR (Deng et al. 2011). In
addition, the involvement of Gαo and Gαi subunits in olfactory signaling was demonstrated in calcium
imaging experiments for D. melanogaster ORs in vitro as well as in vivo. Most interestingly,
interfering with Gαo and Gαi signaling in vivo affected not only tonic and adapting odorant responses,
but also the phasic response, suggesting a direct involvement in the transduction cascade (Ignatious
Raja et al. 2014).
Strong evidence has been provided for the involvement of Gαq-dependent signaling in olfactory
transduction, particularly in moth pheromone transduction (Tab. 6). Remarkably, pheromone
stimulation resulted in a rapid increase of the IP3 concentration in H. virescens, L. migratoria, and
P. americana antennae, which declined within less than 100 ms indicating that pheromone binding
activated Gαq-dependent transduction cascades (Boekhoff et al. 1990a; Boekhoff et al. 1990b; Breer
et al. 1990; Boekhoff et al. 1993; Schleicher et al. 1994). Pheromone/odorant responses of ORNs
were simulated by perfusion with G protein activating fluoride, GTPγS, ATP, specific intracellular Ca2+
concentrations, as well as IP3 (Stengl et al. 1992b; Stengl 1993, 1994; Laue et al. 1997; Wegener et al.
1997; Pophof and Van der Goes van Naters 2002). For M. sexta it was suggested, that pheromone
binding to the receptor activates Gαq, leading to PLCβ-dependent formation of IP3 and DAG. The
IP3-dependent transduction current is composed of a rapid, directly IP3-activated Ca2+ current and a
Ca2+-dependent non-specific cation conductance, which is activated by the influx of Ca2+ (Stengl et al.
1992b; Stengl 1993, 1994). Dependent on the physiological state of the moth, the strength and
duration of the pheromone stimulus, and the resulting amplitude of the Ca2+ inward current a
sequence of different second messenger- and Ca2+-dependent ion channels is activated (Fig. 118,
Zufall and Hatt 1991a; Boekhoff et al. 1993; Schleicher et al. 1994; Reviews: Stengl 2010; Stengl and
Funk 2013). Whether ORCO is involved in one of these transduction currents remains to be
53
Introduction
determined. Consistent with a Gαq-dependent signaling pathway, genetic manipulation of the
involved components severely attenuated odorant responses in D. melanogaster (Kain et al. 2008).
Tab. 6. Metabotropic signaling in insect olfaction (modified after Nakagawa and Vosshall 2009).
Gαo
Insect species
D. melanogaster
B. mori
D. melanogaster
D. melanogaster
D. melanogaster
L. oryzophilus
D. melanogaster
Gαi
D. melanogaster
D. melanogaster
D. melanogaster
Gαs
A. pernyi
D. melanogaster
D. melanogaster
B. mori
A. gambiae
D. melanogaster
M. sexta
D. melanogaster
D. melanogaster
D. melanogaster
Gαq
P. americana
P. americana
P. americana,
L. migratoria
B. mori
M. sexta
H. virescens
D. melanogaster
H. virescens
D. melanogaster
D. melanogaster
B. mori
B. mori,
A. polyphemus,
A. pernyi
L. migratoria
A. polyphemus
M. brassicae
Evidence
Gαo expression in antennae
Gαo expression in antennal nerve bundles
Expression of PeTX affects amplitude and kinetics of odorant responses
Gαo expression in antennae and maxillary palps
Gαo expression in antennae; coexpression of chimeric Gαo improves
odorant responses of heterologously expressed OR22a (HEK 293)
Gαo expression in dendrites of ORNs
Interference with Gαo and Gαi signaling affected odorant responses of
heterologously expressed (HEK 293 cells) ORs and in vivo (phasic, adapted,
and tonic responses)
Gαi expression in antennal ORNs; expression overlaps with Gαq
See above; same results as shown for Gαo
Perfusion with cAMP-analogues or PDE inhibitors increased pheromone
responses
rutabaga (rut) and dunce (dnc) expression in antennae; mutations affected
odorant responses
dnc overexpression resulted in abnormal behavioral odorant responses
Gαs expression in dendrites of ORNs
Gαs expression in olfactory sensilla
ORCO is activated by cyclic nucleotides in heterologous expression
systems
Perfusion with octopamine or 8-Br-cAMP sensitizes pheromone-responses
of ORNs in vivo
Gαs expression in antennal ORNs
Demonstration of cyclic nucleotide sensitivity of ORCO in vivo
Gαs expression in antennae; interference with Gαs signaling severely
affected spontaneous activity and odorant responses in vivo; coexpression
of chimeric Gαs highly improves odorant responses of heterologously
expressed OR22a (HEK293); [35S]GTPγS binding assay provides evidence for
odorant induced G protein activation
Rapid, pheromone-dependent IP3 formation
Rapid IP3 formation dependent on PeTX-sensitive G protein
PIP2 hydrolysis in a pheromone/odorant- and GTP-dependent manner
Reference
(Schmidt et al. 1989)
(Miura et al. 2005)
(Chatterjee et al. 2009)
(Boto et al. 2010)
(Deng et al. 2011)
(Kang et al. 2011)
(Ignatious Raja et al.
2014)
(Boto et al. 2010)
(Deng et al. 2011)
(Ignatious Raja et al.
2014)
(Villet 1978)
(Martin et al. 2001)
(Gomez-Diaz et al. 2004)
(Miura et al. 2005)
(Rützler et al. 2006)
(Wicher et al. 2008)
(Flecke and Stengl 2009;
Flecke et al. 2010)
(Boto et al. 2010)
(Sargsyan et al. 2011;
Getahun et al. 2013)
(Deng et al. 2011)
(Breer et al. 1990)
(Boekhoff et al. 1990a)
(Boekhoff et al. 1990b)
AC1 ion channel is activated PKC-dependently after sustained pheromone
stimulation
Perfusion with GTPγS, ATP, or IP3 resembles pheromone-dependent
currents in ORNs in vitro
Pheromone-induced, rapid IP3 formation; cGMP elevation after strong
pheromone exposure counteracts IP3 formation
rdgB (retinal degeneration B, encodes a phosphatidylinositol transfer
protein) mutation affects odorant response kinetics
Rapid IP3 response is impaired by PKC inhibitors, indicating PKC-dependent
termination of odorant responses
norpA (no receptor potential A) encoded PLC is required for odorant
responses of maxillary palps
Gαq expression in a subset of ORNs
Gαq expression in antennae; perfusion with G protein activating fluoride
simulates pheromone responses
Detection of an IP3 receptor, a Gq protein, calmodulin, and calcineurin in
dendrites of ORNs
(Zufall and Hatt 1991a)
IP3-dependent increase in membrane conductance in ORNs; partly
simulated by odorant stimulation
Identification of PLCβ and PKC in antennal sensilla; pheromone stimulation
increased PKC activity
Gαq expression in olfactory sensilla
(Wegener et al. 1997)
54
(Stengl et al. 1992b;
Stengl 1993, 1994)
(Boekhoff et al. 1993)
(Riesgo-Escovar et al.
1994)
(Schleicher et al. 1994)
(Riesgo-Escovar et al.
1995)
(Talluri et al. 1995)
(Laue et al. 1997)
(Laue and Steinbrecht
1997)
(Maida et al. 2000)
(Jacquin-Joly et al. 2002)
Introduction
Insect species
B. mori
D. melanogaster
B. mori
D. melanogaster
S. littoralis
S. littoralis
D. melanogaster
S. littoralis
D. melanogaster
S. littoralis
D. melanogaster
D. melanogaster
Evidence
Perfusion with sodium fluoride or PKC activators simulated sustained
pheromone stimulation
Gαq-RNAi reduced behavioral responses to a subset of odorants
Gαq expression in ORN dendrites
Overexpression of IP3 kinase1 impairs behavioral odorant responses
A DAG kinase is expressed in antennal trichoid sensilla
Reference
(Pophof and Van der
Goes van Naters 2002)
(Kalidas and Smith 2002)
(Miura et al. 2005)
(Gomez-Diaz et al. 2006)
(Chouquet et al. 2008)
DAG activates a cation channel in ORNs in a PKC-independent manner
Interference with Gαq signaling strongly affects odorant responses, effects
are enhanced by PLCβ-gene or rdgA (retinal degeneration A, encodes a
DAG kinase) mutations, but not by IP3 receptor-gene mutation
A TRP-channel is expressed in olfactory sensilla
See above; same results as shown for Gαi
PLCβ is detected in antennal trichoid sensilla
PKC-dependent phosphorylation of ORCO is required for cyclic nucleotide
sensitivity and is sufficient for ORCO activation in vivo
(Krannich 2008)
(Kain et al. 2008)
(Boto et al. 2010)
(Deng et al. 2011)
(Sargsyan et al. 2011;
Getahun et al. 2013)
1.5.3.7 Temporal control of olfactory sensitivity
1.5.3.7.1 In D. melanogaster the ORNs are the pacemakers controlling olfactory sensitivity
Antennal clocks have been investigated mainly in D. melanogaster, R. maderae, and different
lepidopteran species, whereby most knowledge is available for D. melanogaster: Per-driven
expression of GFP demonstrated per-cycling in the antennae and the proboscis, most probably in
chemosensory cells. It persisted in vitro in DD and was entrainable to a phase-shifted LD cycle. Thus,
it was suggested that ORNs of the antennae and gustatory receptor neurons (GRNs) of the proboscis
are circadian pacemaker cells (Plautz et al. 1997). In subsequent studies, a circadian rhythm was
demonstrated for the sensitivity of the antenna, reflected by the amplitude of electroantennogram
(EAG) responses to stimulation with different odorants (Krishnan et al. 1999; Krishnan et al. 2005).
The rhythm persisted in DD and was abolished in per01 or tim01 mutant flies or in flies solely
expressing per in the central LNvs, indicating regulation by a peripheral circadian clock instead of
central regulation. Indeed, cell specific rescue experiments in per01 mutant flies and disruption of
molecular cycling by expression of dominant negative versions of clk and cyc (clkΔ, cycΔ) clearly
demonstrated that a functional clock in ORNs is necessary and sufficient for rhythms in olfactory
sensitivity. Thus, pacemaker function and physiological output function are colocalized to the same
cell type in the antennal clock, the ORNs (Tanoue et al. 2004). Surprisingly, olfactory sensitivity
peaked in the middle of the night, in the assumed resting phase of the flies (Krishnan et al. 1999). The
phase of the rhythm as well as the independence of the LNvs and their neuropeptide PDF was also
demonstrated on the behavioral level in T-maze experiments (Zhou et al. 2005).
In search of molecules providing mechanisms for rhythmic regulation of olfactory sensitivity, the
function of GPCR kinase 2 (GPRK2) was examined. At least in basiconic sensilla gprk2 is expressed
clock gene-dependently at the same phase as the rhythm of olfactory sensitivity. High levels of
GPRK2 in the middle of the night lead to accumulation of ORs in the ORNs’ dendrites and high
olfactory sensitivity, while low levels of GPRK2 are correlated with low OR expression in the dendrites
and low sensitivity. Thus, GPRK2 is a circadian output protein of the ORN clock, whose rhythmic
expression controls olfactory sensitivity via abundance of ORs (Tanoue et al. 2008).
The rhythm of olfactory sensitivity was not only examined at the antennal level in EAG recordings but
also at the single cell level in single sensillum recordings (SSR), in which electrical activity of the ORNs
55
Introduction
can be recorded and discriminated. Surprisingly, the rhythm was neither reflected in spontaneous
activity nor odorant-evoked spike activity, but in the amplitude of spontaneously generated spikes,
peaking in the middle of the night (Krishnan et al. 2008). This circadian rhythm was shown to be
dependent on the level of gprk2 expression and OR abundance, since it was abolished in orco mutant
flies (Larsson et al. 2004) or Δhalo flies (Dobritsa et al. 2003), lacking the ORs of the recorded ORNs. It
was suggested that rhythmic, GPRK2-dependent OR abundance regulates membrane conductance
and thus rhythms in EAG and spike amplitude (Krishnan et al. 2008).
1.5.3.7.2 Similar to the ORNs, the GRNs control gustatory sensitivity in D. melanogaster
Remarkably, the peripheral clock of the proboscis of fruit flies appears to apply similar mechanisms
as the antennal clock to control gustatory sensitivity (Chatterjee and Hardin 2010; Chatterjee et al.
2010). In this tissue a functional clock in the GRNs was shown to be necessary and sufficient for the
circadian rhythm in spike amplitude, spike duration, and frequency in response to tastants (on the
physiological level), as well as the proboscis extension reflex. Gustatory sensitivity peaks in the
morning and is low in the middle of the night and thus, does not coincide with the highest olfactory
sensitivity, but with the M peak of locomotor activity (see 1.3.4.1) and high mating activity (Sakai and
Ishida 2001; Chatterjee et al. 2010). As shown for the ORN clock, regulation of gustatory sensitivity
also involves rhythmic gprk2 expression and is dependent on the presence of GRs. However, the
function of GPRK2 differs between antennae and proboscis: While in ORNs GPRK2 is expressed in the
same phase like olfactory sensitivity, in GRNs it is expressed in antiphase to gustatory sensitivity.
Thus, contrary to the situation in ORNs, GPRK2 may be involved in GR-dependent downregulation of
gustatory sensitivity in GRNs (Chatterjee et al. 2010). Interestingly, the GRN clock of D. melanogaster
also controls food intake: Flies with non-functional clock in sweet-sensitive GRNs were shown to
consume more food than control flies (Chatterjee et al. 2010).
1.5.3.7.3 In moths and cockroaches the ORNs also appear to be pacemakers
Similar to D. melanogaster, olfactory sensitivity to food odorants as well as pheromones is regulated
by the circadian system in the cockroach R. maderae, as shown by a rhythm in the EAG amplitude
(Page and Koelling 2003; Rymer et al. 2007; Saifullah and Page 2009) and the odorant-evoked spike
frequency measured in single sensillum recordings (Saifullah and Page 2009). Surprisingly, the
rhythm peaks in the early subjective day (around dawn) and thus, shows a different phase than
locomotor activity and mating activity, both peaking around subjective dusk (Rymer et al. 2007).
Since the rhythm in the EAG amplitude was abolished, when the optic tracts of the experimental
cockroaches were bilaterally sectioned (i.e. the optic lobes including AME were isolated), it was
concluded that the central pacemaker controls antennal sensitivity (Page and Koelling 2003).
However, the rhythm in spike frequency, measured on the single cell level, persisted in animals with
isolated optic lobes, indicating the presence of a pacemaker outside the optic lobes regulating ORN
sensitivity (Saifullah and Page 2009). Whether the responsible pacemaker is localized to the ORNs
itself, as shown for D. melanogaster, remains to be examined. The loss of rhythmicity on the
antennal level was suggested to be a result of a fast desynchronization of the ORNs in the absence of
the central pacemaker (Saifullah and Page 2009).
56
Introduction
Substantial knowledge is also available for the situation in moths and other lepidopteran species:
Since pheromone production and calling behavior of female moths, male upwind flight, behavioral
pheromone responses, and of course mating activity is regulated by the circadian clock and
synchronized to a time window in the scotophase (Lingren et al. 1977; Baker and Cardé 1979; Sasaki
and Riddiford 1984; Itagaki and Conner 1988; Linn et al. 1996; Rosen 2002; Rosen et al. 2003;
Silvegren et al. 2005), it seems reasonable that the male's antennal sensitivity to pheromone
components is also under circadian control. In studies performed on T. ni (Payne et al. 1970),
Choristoneura fumiferana (Worster and Seabrook 1989) and Agrotis segetum (Rosen et al. 2003) no
rhythmic changes in the EAG amplitude in response to pheromones were detected, and it was
suggested that pheromone sensitivity is regulated by central mechanisms. However, other studies
found evidence for the presence of antennal clocks and regulation of sensitivity at the periphery:
Clock genes are expressed in chemosensory cells of antennae, legs, and proboscis of Mamestra
brassicae (Merlin et al. 2006) and molecular oscillations were demonstrated for antennae of Bombyx
mori (Iwai et al. 2006), Spodoptera littoralis (Merlin et al. 2007), and Danaus plexippus (Merlin et al.
2009), which persisted in DD (Merlin et al. 2007; Merlin et al. 2009) and were entrainable to other LD
cycles (Merlin et al. 2009). Astonishingly, the antennal clock of D. plexippus appears to be involved in
time-compensated sun compass navigation, a function that was not expected previously from the
antenna (Merlin et al. 2009). In EAG recordings circadian changes of pheromone sensitivity were
shown for S. littoralis, which surprisingly decreased around dawn (Merlin et al. 2007), when
behavioral responsiveness to the pheromone blend is highest (Silvegren et al. 2005). In search for
circadian output molecules rhythmic expression could be demonstrated for a putative odorantdegrading enzyme, but not for pheromone binding protein 1 or orco (Merlin et al. 2007). In the
hawkmoth M. sexta different antennal cell types (including virtually all ORNs and supporting cells of
all sensilla types) express per. Most probably, the expression level is higher in the evening as
compared to the morning (Schuckel et al. 2007). On the physiological level tip recordings of trichoid
sensilla revealed daytime-dependent changes in pheromone sensitivity and temporal dynamics of
the pheromone response, showing more tonic responses with decreased spike frequency in the
middle of the day (ZT 8 - 11, Flecke et al. 2006; Flecke and Stengl 2009).
1.5.3.8 Pheromones and general odorants are Zeitgeber for entrainment
As mentioned before, social cues are also prominent Zeitgeber for entrainment. When the genotypic
composition of a group of D. melanogaster was manipulated, clock gene cycling in the oenocytes and
the head, pheromone display on the cuticle, as well as mating was affected. Thus, the social context
affects central and peripheral clock mechanisms (Krupp et al. 2008). Social cues have been shown to
improve synchrony between individual flies and interestingly, these cues appear to be olfactory cues.
Entrainment by social cues depends on functional peripheral clocks, most probably in the antennae
(Levine et al. 2002a; Lone and Sharma 2011). The odorant receptor OR47b was identified to be
crucial for socio-sexual interactions, and OR65a and OR88a appear to play minor roles, while the cVA
receptor OR67d is not required (Lone and Sharma 2012).
Pheromones may as well function as Zeitgeber for phase-synchronization of high mating preference
of females and high pheromone sensitivity of males, as shown for the moth S. littoralis (Silvegren et
al. 2005). For this species synchrony between both sexes was shown to be extremely important,
57
Introduction
since no mating occurred, when male and female S. littoralis were entrained ten hours out of phase
(Silvegren et al. 2005). However, other species seem to be more flexible in this respect: R. maderae
for example exhibited mating rhythms with two peaks, each corresponding to the phase of one sex,
when males and females were entrained in antiphase (Rymer et al. 2007).
1.5.3.9 Phase-discrepancies between olfactory sensitivity, mating, and
locomotor activity
While the occurrence of mating in R. maderae was shown to be clearly dependent on the presence of
the antennae (Rymer et al. 2007), in D. melanogaster cuticular hydrocarbons, which function as
pheromones involved in courtship and mating, are detected by GRs expressed in gustatory organs
(Bray and Amrein 2003; Lacaille et al. 2007; Reviews: Ferveur 2005; Ebbs and Amrein 2007).
Consistently, high gustatory sensitivity in the morning (at least when measured in the proboscis)
coincides with high mating activity (Sakai and Ishida 2001; Chatterjee et al. 2010). However, the
abundance of cuticular hydrocarbons generally appears to be lower during the subjective day and
higher in the subjective night, thus not coinciding with high gustatory responsiveness (Krupp et al.
2008). As mentioned above, such phase discrepancies were also found for the antennal clock of
several species: In D. melanogaster antennal as well as behavioral responsiveness peaks in the fly’s
resting phase (Krishnan et al. 1999; Zhou et al. 2005). In R. maderae high pheromone responsiveness
coincides with low mating activity and low locomotor activity (Page and Koelling 2003; Rymer et al.
2007). In S. littoralis antennal pheromone responsiveness is low when behavioral responsiveness is
high (Silvegren et al. 2005; Merlin et al. 2007). For D. melanogaster it was suggested, that high
olfactory sensitivity during the resting phase may increase the probability to escape from dangerous
situations while resting and thus may increase survivability (Krishnan et al. 1999; Zhou et al. 2005).
Another explanation was provided by Schendzielorz et al. (2012), who investigated antennal levels of
cyclic nucleotides in R. maderae and found antiphasic, daytime-dependent oscillations for cAMP and
cGMP. Moreover, cGMP levels were increased pheromone-dependently, while cAMP levels were
decreased pheromone-dependently and increased OA-dependently, consistently with their roles in
adaptation and sensitization (see 1.5.3.7). It was suggested that stress and overstimulation with
pheromones as well as other odorants under laboratory raring conditions may modulate olfactory
sensitivity cyclic nucleotide-dependently, which may mask the phase and thus may be responsible for
the observed phase differences (Schendzielorz et al. 2012).
1.6 Aim of this thesis
The work of my thesis aimed at the physiological characterization and comparison of central and
peripheral insect circadian pacemaker cells. AME neurons of the Madeira cockroach R. maderae were
used as model system for central clock neurons, since the AME is particularly well characterized and
primary cell cultures of these cells were available (Petri and Stengl 1999; Review: Homberg et al.
2003). Among the variety of peripheral clocks, the antennal clock of insects, which is located in the
ORNs and regulates olfactory sensitivity, is best characterized. Although ORN clocks have been
58
Introduction
reported for R. maderae, ORNs of the hawkmoth M. sexta were chosen as model system, due to the
more precise characterization of olfactory transduction in moths as well as the availability of primary
cell cultures (Stengl and Hildebrand 1990; Review: Stengl 2010).
The central AME neurons express the core feedback loop genes in a circadian manner (Werckenthin
et al. 2012) and generate rhythms in their electrical activity with different period lengths, ranging
from circadian rhythms to ultradian rhythms in the millisecond range, allowing for very regular
spontaneous activity, which is a prerequisite for temporal encoding (Schneider and Stengl 2005,
2006, 2007). Apparently these rhythms are synchronized by different coupling interactions, such as
neuropeptide signaling, with PDF being the most important coupling factor within the central clock
as well as between the central clock and peripheral clocks, as shown for the oenocyte clock of
D. melanogaster, which regulates pheromone synthesis (Schneider and Stengl 2005; Krupp et al.
2013; Review: Taghert and Nitabach 2012). So far, the AME neurons of R. maderae have been
investigated in extracellular recordings of the isolated AME (Schneider and Stengl 2005, 2006, 2007)
and in Ca2+ imaging experiments (Wei and Stengl 2012; Baz et al. 2013). In these studies first Ca2+dependent ion channels were proposed to be involved in the regulation of spontaneous activity (Wei
and Stengl 2012), and it was started to characterize the effects of PDF on these neurons (Schneider
and Stengl 2005). In D. melanogaster the PDF receptor was cloned and shown to be a class II peptide
GPCR, which couples to Gαs and probably also to Gαq (Mertens et al. 2005; Shafer et al. 2008; Agrawal
et al. 2013), but only limited information is available for the effects of acute PDF application on
central pacemaker neurons (Seluzicki et al. 2014). In this study, extracellular recordings of the
isolated AME and patch clamp experiments on single AME neurons were performed. These
experiments aimed at a detailed characterization of the physiological properties and the expressed
ion channels of these neurons. Additionally, the PDF-dependent signal transduction cascade and ion
channels involved therein were investigated, both at the network and the single cell level.
Despite some differences in the molecular clockwork, peripheral clocks were suggested to employ
similar mechanisms as central clocks and to be synchronized by signals from central clocks as well as
external Zeitgebers. Remarkably, in the case of the peripheral ORN clock, pheromones and general
odorants also appear to function as synchronization factors, as shown for fruit flies and moths
(Levine et al. 2002a; Silvegren et al. 2005). One central topic in insect olfaction during the last years
was the function of the olfactory coreceptor ORCO (Review: Nakagawa and Vosshall 2009). While
ORCO expression was shown in ORNs of M. sexta (Patch et al. 2009; Grosse-Wilde et al. 2010;
Grosse-Wilde et al. 2011), no functional characterization has been performed so far. Based on
research performed in other insects, it was hypothesized that ORCO could function as a pacemaker
channel in the ORN clock of M. sexta, which might drive SMPOs and spontaneous activity as a basis
for temporal encoding, instead of mediating odorant-induced ionotropic signal transduction in vivo
(Stengl 2010). In this thesis, the function of M. sexta ORCO (MsexORCO) was characterized in a
heterologous expression system. Therefore HEK 293 cells were transiently transfected with
MsexOrco and putative pheromone receptors and the expression of MsexORCO was
immunocytochemically examined. Ca2+ imaging experiments were employed to investigate, whether
MsexORCO functions as ion channel and how it might be regulated. Additionally, the effects of
different ORCO agonists and antagonists were investigated in primary cell cultures of M. sexta ORNs.
59
60
Material and methods
2 Material and methods
2.1 Animal rearing
2.1.1 Cockroach rearing
The Madeira cockroach (Rhyparobia maderae (Fabricius 1781), also well known under its synonym
Leucophaea maderae) is a member of the family Blaberidae (subfamily Oxyhaloinae, Fig. 19). It is an
ovoviviparous species, retaining the eggs in the mother's body until the embryos are fully developed
and ready to hatch. The development from larvae to imagines depends on sex and environmental
conditions and takes more than four months for males and nearly six months for females, during
which the larvae pass through seven (male) or eight (female) instars. Imagines reach a size of 4 - 5 cm
(Cornwell 1968).
Breeding colonies were first kept at the Philipps University of Marburg and later at the University of
Kassel. Several hundred animals were maintained in plastic boxes (60 cm x 40 cm x 40 cm) at 25 °C
and 50 % relative humidity in 12:12 h light:dark cycles. The boxes contained litter for rodents and a
stack of egg cartons giving an opportunity to hide. Cockroaches were provided with dry dog food
(Happy Dog, Interquell GmbH, Wehringen, Germany), potatoes, apples, and cucumber at least two
times a week and had water ad libitum.
Fig. 19. The Madeira cockroach Rhyparobia maderae. A, B. Dorsal (A) and ventral view (B) of a male individual. Scale bar:
10 mm. The circle in B marks the detail that is magnified in C. C. The last abdominal segment shows two types of
appendices, cerci (arrowheads) and styli (arrows). Since only male cockroaches have styli, these appendices can be used as
distinctive mark. Photos by Achim Werckenthin, figure modified after (Werckenthin 2013).
61
2.1.2 Moth rearing
The hawkmoth Manduca sexta (Linnaeus 1763) is a member of the family Sphingidae (subfamily:
Sphinginae) with widespread occurrence in North and South America. The blue-green larvae (tobacco
hornworm) reach a size of approximately 7 cm and the imagines achieve a wing span of
approximately 10 cm (Fig. 20). M. sexta's developmental cycle takes approximately 40 days, during
which the larvae pass through 5 instars.
Animals used for the preparation of primary ORN cell cultures were bred in cultures of the University
of Kassel in L:D cycles of 17:7 h at 24 - 27 °C and 40 - 60 % relative humidity. Imagines were kept in
cages containing a tobacco plant (Nicotiana attenuata) for oviposition and artificial flowers
containing a 1.5 ml microcentrifuge tube with sugar solution for nutrition (Riffell et al. 2008). An
artificial flower scent was added to the artificial flowers, supporting the identification as feeding
source. Eggs were collected from the tobacco plant and transferred to plastic boxes containing an
artificial diet modified after (Bell and Joachim 1976). Larvae were consecutively transferred to bigger
plastic boxes containing the same diet. At the end of the 5th larval instar, when the dorsal heart
becomes visible (= onset of wandering), larvae were separated in wooden tubes or boxes filled with
litter allowing for pupation. Hawkmoths used for molecular cloning were bred in cultures in the Max
Planck Institute for Chemical Ecology (Jena) in L:D cycles of 16:8 h, as described by Grosse-Wilde et
al. (2011). Eggs and larvae were kept at 27 °C and 70 % relative humidity. 5th instar larvae were
separated in boxes filled with crumpled paper allowing for pupation. Approximately one week before
emergence the pupae were transferred to paper bags and allowed to emerge at 25 °C and 50 %
relative humidity.
Fig. 20. The Hawkmoth Manduca sexta. A. Eggs (arrow), 5th instar larva, pupa, and adult, male M. sexta, which has
broader antennae compared to the female. Eggs have a diameter of 1 - 1.5 mm. B,C. The sexes of the pupae can be
distinguished on the basis of the genital aperture, located on segment 8 in female pupae (arrow, B) and on segment 9 in
male pupae (arrow, C). The anus (arrowhead) is located on segment 10 in both sexes. Photos by Achim Werckenthin. Scale
bar: 3 mm.
62
2.2 Cloning of M. sexta or- and snmp-1 genes
Cloning of M. sexta genes was performed by Ewald Grosse-Wilde and Sascha Bucks (Max Planck
Institute for Chemical Ecology, Jena) as described in (Nolte et al. 2013): "The odorant receptors
(MsexOR-1, MsexOR-4), MsexOrco (= MsexOR-2) and sensory neuron membrane protein 1
(MsexSNMP-1) were identified previously (Patch et al. 2009; Grosse-Wilde et al. 2010; Grosse-Wilde
et al. 2011)." … "Antennal mRNA was reverse transcribed using the Superscript II Mix (Invitrogen, Life
Technologies, Carlsbad, CA, USA). The resulting cDNA was used as template to amplify respective
coding sequences for MsexOrco, MsexOR-1, MsexOR-4 and MsexSNMP-1 via PCR, adding suitable
restriction sites outside the coding sequence (for primers see 5.2). PCR products were cloned into
pCR®II-TOPO® vectors and transformed into E. coli (One Shot® Top10 competent cells, Invitrogen).
Plasmids were isolated (QIAprep Spin MiniPrep Kit) and treated with suitable restriction enzymes
(NEB, Ipswich, MA). Fragments were gel-purified and subcloned into the pcDNA3.1(-) expression
vector (Invitrogen). After further replication in E. coli the vector was isolated (QIAGEN Plasmid Maxi
Kit) and stored at -20 °C."
DmelOrco was generously provided by Prof. Dr. Hans Hatt (Ruhr-University Bochum, Germany).
DmelSnmp-1 was cloned and provided by Jackson Sparks and Prof. Dr. Richard G. Vogt (University of
South Carolina, USA) and Dr. Jing-Jiang Zhou (Rothamsted Research, Harpenden, UK). The murine Gprotein α-subunit Gα15 was a kind gift by Dr. Jürgen Krieger (University of Hohenheim, Germany). All
sequences were inserted in pcDNA3.1 expression vectors.
2.3 Cell culture
2.3.1 Preparation of culture dishes
Primary cell cultures: Round coverslips with a diameter of 8 mm (Menzel Gläser, now part of Thermo
Fisher Scientific, Braunschweig, Germany) were sterilized by incubation in 100 % ethanol for 10 min
and subsequent short flame-scarfing. Each coverslip was placed in a sterile tissue culture dish (35 mm
diameter, Greiner Bio One, Frickenhausen, Germany). Then 30 µl coating solution, containing
164 µg/ml concanavalin A (Sigma-Aldrich) and 14 µg/ml poly-D-lysine (Sigma-Aldrich), was added
onto each coverslip. After an incubation time of 2 h at room temperature (RT) the coating solution
was removed and the coverslips were washed twice with sterile bidestilled water (ddH2O).
Alternatively, the coverslips were incubated in a coating solution solely containing concanavalin A
(200 µg/ml) for 1 h (personal communication with Dr. Petra Schulte, Forschungszentrum Jülich). All
steps were performed at a sterile bench (HERA guard, Heraeus, Hanau, Germany). Culture dishes
with coated coverslips were transferred into a closed glass dish and stored at 4°C.
Human embryonic kidney (HEK 293) and Spodoptera frugiperda (SF9) cell culture: Lids of 35 mmdishes or round 12 mm-coverslips (Carl Roth, Karlsruhe, Germany) were coated by incubation in
0.01 % poly-L-lysine solution (Sigma-Aldrich) for 10 min at RT followed by washing in autoclaved
ddH2O and UV-irradiation for 20 minutes.
63
2.3.2 Preparation of cell culture media
All cell culture media were adjusted to pH 7.0 and an osmolarity of 380 mosm/l (OM 806 osmometer,
Löser, Berlin, Germany) with D-mannitol (Sigma-Aldrich), if not indicated otherwise. The composition
of the solutions is listed in the appendix (see 5.3). After preparation all solutions were sterile-filtered
(pore size: 0.22 µm, Merck Millipore, Darmstadt, Germany). Subsequently the flasks were stored at
4 °C and only opened under sterile bench conditions.
2.3.3 Primary AME cell cultures
Primary cell cultures of neurons associated with the accessory medulla (AME) of R. maderae were
obtained and kept as described in (Petri and Stengl 1999): Adult male cockroaches were anesthetized
in iced water, surface sterilized with Barrycidal solution (Interchem AG, Zug, Switzerland), and
decapitated. For dissection the head capsule was pinned on wax, opened with a razor blade breaker,
and covered with dissection medium. Fat body, tracheae, and neurolemma were carefully removed
with Dumont #5 precision forceps to uncover the optic lobes. The AME tissue with associated
neurons was excised with borosilicate glass capillaries (GC150T-10; Clark Electromedical Instruments,
Reading, UK), which were pulled with a P 87 or P 97 microelectrode puller (Sutter Instruments,
Novato, CA, USA) and manually broken to obtain a tip diameter of approximately 200 µm. The AME
was localized on the basis of its position at the ventromedial edge of the medulla (Fig. 21). Both
AMAE were transferred into a low bind reaction cup (Biozym Scientific GmbH, Hessisch Oldendorf,
Germany) and incubated in 500 µl dissociation medium containing 1 mg/ml papain (crude powder,
Sigma-Aldrich) at 37 °C, allowing for dissociation of the cells. All subsequent steps were performed at
a sterile bench (HERA guard, Heraeus). The enzymatic dissociation was terminated after 6 min by
washing the tissue three times with cold washing medium. It was always tried to prevent
centrifugation to protect the cells, but in cases, where the tissue was floating at the surface of the
fluid, it was required to spin down the tissue for 2 min at 2000 rpm (Minispin, Eppendorf, Hamburg,
Germany). After washing the tissue was gently triturated in 100 µl culture medium 1 (containing 5 %
fetal bovine serum, FBS) using an Eppendorf-pipette assembled with 100 µl low bind tip (Biozym,
Hessisch Oldendorf, Germany). The cell suspension (50 µl) was added onto a coated coverslip located
in culture dishes (2.3.1) and the cells were allowed to adhere for 2-4 h. Then, 1.5 ml culture medium
1 was added. The cultures were transferred into a closed glass dish, kept at 20 °C in a self-made
incubator (Privileg fridge, modified by the workshop of the University of Regensburg). Several waterfilled, open culture dishes were added into the glass dish, to enhance relative humidity. After one day
the complete culture medium was changed. Subsequently, 50 % of the medium was substituted once
or twice per week.
64
Fig. 21. Protocerebrum of the
cockroach R. maderae. For better
accessibility fat body and tracheae
were removed. The circle marks
the area containing the AME and
its associated neurons of the left
optic lobe, which was punched out
with a glass capillary. AL: antennal
lobe, AN: antennal nerve, CE:
complex
eye,
CP:
central
protocerebrum, LA: lamina, LO:
lobula, ME: medulla. Photo by
Thomas Reischig (University of
Marburg).
Alternatively, a different method was employed, which was developed by Dr. Petra Schulte
(Forschungszentrum Jülich) for TAG neurons of G. bimaculatus and slightly modified: After dissection
the AMAE were transferred into a low bind reaction cup filled with 400 µl of dissociation medium 2
containing 1 mg/ml collagenase (Sigma-Aldrich) and 4 mg/ml dispase (Sigma-Aldrich). After an
incubation of 2.5 min at 37 °C the tissue was gently triturated with an Eppendorf-pipette assembled
with a 100 µl low bind tip. To terminate the enzymatic dissociation the content of the cup was
transferred to a 15 ml-falcon tube (Greiner Bio One) containing 10 ml cold washing medium 2. After
centrifugation at 8 °C and 500 rpm for 5 min the supernatant was discarded and the cells were gently
resuspended in 100 µl culture medium 2, which did not contain FBS but was supplemented with
glucose, fructose, L-proline, and imidazole. The cell suspension was distributed between two culture
dishes as described above.
With the aim of improving the AME cell cultures both protocols as well as combinations thereof were
applied. Additionally other modifications of the culture protocol were tested:




Instead of using random adult, male cockroaches, cockroaches were sacrificed around one
day after adult ecdysis, since the tissue should loosen before ecdysis and thus be more
suitable for dissociation.
It was expected, that the procedure of searching the colony for male cockroaches and
catching them causes stress for the animals. Since a negative impact of the stress on the
condition of the cells could not be excluded, the cockroaches were given the opportunity to
calm down after catching. Therefore the cockroaches were placed in a small vessel with a
hiding place for approximately 30 min, from which they were directly anesthetized with iced
water before dissection.
Since a preceding incubation in EGTA-solution proved beneficial for the dissociation of other
insect nervous tissues (Stengl and Hildebrand 1990), the AMAE were incubated occasionally
in 5 mg/ml ethylene glycol tetraacetic acid (EGTA) as described in 2.3.4.
Different variations of the papain-dependent dissociation-procedure were tested: Different
durations for the incubation at 37 °C were tested (4 - 12 min). Since papain was reported to
be activated by cysteine (Klein and Kirsch 1969), the papain solution was occasionally
65







supplemented with 0.2 mg/ml L-cysteine (Sigma-Aldrich), and incubations were performed at
37 °C or at RT.
L-glutamine is an essential nutrient in cell cultures, which is reported to exhibit a
spontaneous degradation, whereas the cell-toxic ammonia is produced (Tritsch and Moore
1962). Therefore L-15 medium containing L-glutamine (PAA, part of GE Healthcare, Cölbe,
Germany) was substituted for L-15 medium without L-glutamine (PAA), and only smaller
aliquots were supplemented with L-glutamine (Invitrogen). Occasionally L-glutamine was
substituted with GlutaMAX™ (Invitrogen), a dipeptide (L-alanine-L-glutamine), which is more
stable in aqueous solutions than L-glutamine. L-glutamine is continuously released after
hydrolysis of GlutaMAX™ by peptidases, which are released by the cells, thereby providing a
regenerating pool of L-glutamine. However, GlutaMAX™ is only recommended for
mammalian cell culture.
It could not be excluded, that an unknown part of AME-cells was lost due to attachment to
the wall of the reaction cup or the pipette tip, when the tissue is triturated. Therefore
standard microcentrifuge tubes and pipette tips were substituted for low bind cups and tips
(Biozym). In some experiments the trituration was performed with a fire-polished pasteur
pipette, to prevent the potential loss of cells. However, most triturations were performed
with Eppendorf pipettes due to better handling properties.
Initially all cell culture media were adjusted to pH 7.0 and an osmolarity of 360 mosm/l (Petri
and Stengl 1999). Since the electrophysiological experiments were performed in ringer
solution, adjusted to pH 7.1 and 380 mosm/l, which allowed for extracellular recordings from
isolated AMAE for several days, these adjustments were also transferred to the cell culture
media.
The supplements of the culture medium were changed: Usually the culture medium was L-15
with 5 % FBS. Since FBS might differ considerably from batch to batch, standard FBS was
substituted for FBS Gold (PAA), whose composition is defined and not batch-dependent.
Since FBS might contain substances that are harmful for certain cells, it was tested to forego
the use of FBS with the use culture medium 2, which instead was supplemented with
different nutrients and buffer substances (supplemental mix, 5.3.1). Additionally it was
tested to supplement the culture medium with the supernatant of a non-neuronal M. sexta
cell line, which highly improved the culture conditions for M. sexta ORNs (Stengl and
Hildebrand 1990, 2.3.4, 2.3.5).
Two different coatings of the coverslips were employed: The coverslips were coated with
poly-D-lysine and concanavalin A (Petri and Stengl 1999) or solely with concanavalin A
(personal communication with Dr. Petra Schulte, Forschungszentrum Jülich).
Two different temperatures were used to maintain the cell cultures: A low temperature of
20 °C was chosen, since it allowed for long-term AME cultures in a previous study (Petri and
Stengl 1999) and for long-term M. sexta ORN cultures (Stengl 1993). Since other studies
suggested higher temperatures between 26 °C and 30 °C (Kreissl and Bicker 1992; Cayre et al.
1998; Gocht et al. 2009) and the cockroaches were also kept at a temperature of 27 °C, it was
tested to maintain the cell cultures at 27 °C.
The culture medium was exchanged either completely (to prevent a continuous increase of
the osmolarity) or partly (around 50 %, to facilitate removal of toxic substances and debris
but nevertheless retain a certain amount of positive factors in the culture).
66
2.3.4 Primary M. sexta ORN cell cultures
Primary cell cultures of M. sexta ORNs were prepared by Christa Uthof, Hongying Wei, and El-Sayed
Baz (University of Kassel), following a protocol modified after (Stengl and Hildebrand 1990). Pupae of
M. sexta were staged according to a timetable, based on unpublished observations of Mary M.
Nijhout (Harward University) in the 1970s (Jindra et al. 1997). Male pupae (late stage 2 - stage 3)
were anesthetized with iced water, surface sterilized with Barrycidal solution, and dissected in
supplemented HBSS (sHBSS). Isolated antennal tubes were washed in sHBSS and subsequently
incubated in sHBSS containing 5 mg/ml EGTA for 5 min at 37 °C. The enzymatic dissociation was
performed in dissociation medium 1 containing 1 mg/ml papain in two batches for maximum 20 min
at 37°C and terminated by adding washing medium 1. After centrifugation for 8 min at 1000 rpm the
supernatant was discarded and the cells were resuspended in sHBSS. The cell suspension was
distributed between concanavalin A/poly-D-lysine-coated coverslips placed in tissue culture dishes.
The cells were allowed to settle for 30 min, before 1 ml of 2:1 medium (consisting of two parts L-15
with 5 % FBS and one part conditioned medium) was added to each culture dish. After 1 day the 2:1
medium was exchanged completely. Subsequently about 50 % of the medium was replaced at least
once per week. The cultures (Fig. 22) were kept as described in 2.3.3.
Fig. 22. Olfactory receptor neurons of M. sexta in primary cell
culture. Antennal tissue of a M. sexta pupa was dissociated to
obtain a primary cell culture containing different types of cells.
Putative olfactory receptor neurons are indicated by arrows and
different non-neuronal cells by arrowheads. The cells were loaded
with the calcium indicator fura-2. Fluorescence was detected at
530 nm after excitation at 380 nm.
2.3.5 M. sexta MRRL-CH1 cell culture
The MRRL-CH1 cell line is a non-neuronal cell line that was obtained from M. sexta embryonic tissue
(Eide et al. 1975). It was a gift from Dr. Dwight E. Lynn (formerly University of Arizona) in the 1980s
and has been kept in culture since then. At the University of Kassel the cell line has been maintained
by Christa Uthof.
The cells were kept in 50 ml culture flasks (Becton, Dickinson and company, Franklin Lakes, NJ, USA)
containing 10 ml cell line nutritive medium (CLNM) in an incubator at 20°C. In intervals of six weeks
the supernatant (= conditioned medium) was collected and stored at -80 °C. For the generation of
67
subcultures 5 ml CLNM were added into the flasks, the cells were gently suspended with a pasteur
pipette, and transferred into new culture flasks containing CLNM.
2.3.6 HEK 293 cell culture
HEK 293 cells were obtained from the Leibniz Institute DSMZ - German Collection of Microorganisms
and Cell Cultures (Braunschweig, Germany). Maintenance and transfection of the cells were
performed by Sabine Kaltofen or Sylke Dietel-Gläßer. Cells were grown in a 1:1 mixture of Dulbecco’s
Modified Eagle Medium and Ham’s F-12 medium with L-glutamine (DMEM/Ham’s F-12, PAA, Cölbe,
Germany) containing 10 % FBS (PAA) in T-75 flasks (Greiner Bio One) at 37°C and 5 % CO2 content
(Incubator: HERAcell 150, Heraeus, Hanau, Germany). At a density of 1.0 - 1.5*107 cells were splitted
into new culture flasks (= passaging). For this purpose cells were washed one time with HBSS (PAA)
and separated by incubation in 5 ml HBSS containing 0.5 g/l trypsin and 0.2 g/l
ethylenediaminetetraacetic acid (EDTA) for 2 min at RT. The reaction was terminated by adding 5 ml
culture medium and subsequent centrifugation. Cells were resuspended and used for seeding new
culture flasks at densities of 2*105 - 7*105 depending on the expected time of next passaging.
Passages 4 to 16 were used for experiments.
The cells were plated on coated coverslips or lids of 35 mm-culture dishes (Greiner Bio One) at a
density of 105 cells per 1.5 ml medium per lid or per dish containing four coverslips. About 24 h after
plating cells were transfected using Roti®-Fect transfection reagent (Carl Roth) according to
instructions. An amount of 1 µg DNA and 5 µl Roti®-Fect were diluted in 60 µl FBS-free medium. Both
solutions were combined and incubated for 30 minutes allowing for formation of nucleic acid-lipid
complexes, which are taken up by the cells. After incubation the solution was applied to the
respective culture containing 1.5 ml medium. In some cases cells were transfected using XtremeGENE HP DNA transfection reagent (Roche). For this purpose 1.5 µg DNA and 3 µl transfection
reagent were diluted in 150 µl serum-free medium, incubated for 30 minutes and applied to the
culture. Cells were used 2 - 4 days after transfection for experiments.
Fig. 23. Human embryonic kidney
cells (HEK 293) in culture. A. HEK
293 cells at approximately 80 %
confluence monitored with phase
contrast optics (photographed by
Sabine Kaltofen). B. HEK 293 cells
monitored at higher magnification.
The image was focus-stacked after
acquisition. Scale bar: 20 µm.
68
2.3.7 SF9 cell culture
SF9 cells were also obtained from the Leibniz Institute DSMZ. Maintenance and transfection of the
cells were performed by Sabine Kaltofen or Sylke Dietel-Gläßer. The cells were grown at 26 °C
without CO2 addition in T-75 flasks (Greiner Bio One) containing SF-900 III SFM medium (Invitrogen)
without further supplements. After seven days, when the cells were grown to 90 % confluence (23*107 cells), the cultures were split. The old medium was collected and centrifuged to separate the
debris. The cells were resuspended with a cell scraper in 10 ml fresh medium and seeded at densities
of 3 * 106 and 5*106 cell in new T-75 flasks containing 10 - 12 ml fresh medium and 3 - 5 ml
supernatant of the old medium. For calcium imaging experiments the cells were seeded in coated lids
of 35 mm-culture dishes (Greiner Bio One) at a density of 1 - 2*106 cells depending on the scheduled
use. The medium was composed of 1 ml fresh medium and 0.5 ml supernatant of the old medium.
Transfection of the cells with plasmid DNA was performed about 24 h after plating using Cellfectin II
(Invitrogen) transfection reagent. For each cell culture 8 µl transfection reagent and 1 µg DNA were
dissolved in 100 µl SF-900 III SFM medium. Both mixtures were combined, vortexed, and incubated
for 30 min at RT allowing for the formation of lipid-DNA-complexes. After incubation the solution was
gently added to the culture dish. The medium was not changed until experiments were performed
after 2 - 4 days.
Fig. 24. Spodoptera frugiperda (SF9) cells in culture. SF9 cells are regular
in size (about 20 µm cell diameter) and morphology (spherical). The cells
were loaded with the calcium indicator fura-2. Fluorescence was detected
at 530 nm after excitation at 380 nm.
2.4 Immunocytochemistry
Immunocytochemical experiments were performed on transiently transfected HEK 293 cells, to
examine the expression of the transfected DNA. HEK 293 cells were grown on poly-L-lysine-coated
coverslips in 24-well plates (Greiner Bio One). Approximately 48 h after transfection cells were
washed for 5 min in phosphate-buffered saline (PBS). After fixation in PBS containing 4 %
paraformaldehyde for 1 min, cells were washed in HBSS (three times, each 5 min) and incubated in
HBSS containing Texas Red®-conjugated wheat germ agglutinin (WGA, 25 µg/ml, Invitrogen) for
10 minutes. Next, cells were washed again in PBS (three times, each 5 min) followed by
permeabilization and blocking of unspecific binding sites in blocking buffer (PBS containing 0.1 %
Triton X-100 and 5 % normal goat serum) for 60 minutes. The cells were incubated overnight with the
primary antibody (anti-BmorOrco or anit-BmorOR-3 at a dilution of 1:500 or 1:1000 in blocking
buffer) at 4°C. On the next day cells were washed in PBS (five times, each 5 min) followed by
69
incubation with the secondary antibody (Alexa Fluor® 488 conjugated goat anti rabbit
immunoglobulin G (IgG, Invitrogen) at a dilution of 1:1000 in blocking buffer) for 60 min. Finally cells
were washed again in PBS (five times, each 5 min), mounted in Vectashield medium (Vector
Laboratories, Burlingame, CA) and sealed with nail polish.
Some of the preparations were opened afterwards to perform a nuclear staining. For this purpose
cells were washed in PBS (three times, each 5 min) and incubated in PBS containing 4'-6-diamidino-2phenylindole (DAPI 5 µg/ml, Carl Roth) for 15 min. After washing (three times, each 5 min), cells were
mounted in PBS containing 33 % glycerol (Carl Roth) and sealed with Entellan (Merck). If not
indicated otherwise, all washing and incubation steps were performed at room temperature on a
rotator.
Both primary antibodies were a kind gift from Dr. Jürgen Krieger (Hohenheim, Germany). AntiBmorOrco was raised against the peptide NQSNSHPLFTESDARYH and anti-BmorOR-3 against the
peptide KDNSEYAMKTHRRVHK (Fig. 25, Fig. 26).
Fluorescence images were acquired using a confocal microscope system (Leica TCS SP5 II) with a 20
fold magnification water immersion objective (Leica HCX PL APO) controlled by Leica LAS AF software
(version 2.5.1.6757). DAPI fluorescence was excited at 405 nm with an UV-laser and emission was
detected between 450 and 470 nm. Alexa Fluor® 488 was excited at 496 nm employing an argon
laser and emission was detected in a range from 509 to 529 nm. Texas Red®-X was excited at 543 nm
using a helium neon laser and emission was detected between 595 and 615 nm.
MsexORCO:
MTMLLRKMYSTVHAILIFVQFVCMGVNMAMYADEVNELTANTITVLFFAHSIIKLGFLAFTSKSFYRTMAVWNQSNSHPLFTESDARYHQIALT
KMRRLTYFICFMTVMSVVSWVTITFFGESVRMIANKETNETLTEPAPRLPLKAWYPFDTMSGSMYVFVFVFQIYWLLFSMSMANLLDVLFCAWL
IFACEQLQHLKAIMKPLMELSASLDTYRPNTAELFRVSSTDKSEKVPDPVDMDIRGIYSTQQDFGMTLRGTGGKLQNFVQNTVNPNGLTQKQEM
LARSAIKYWVERHKHVVRLVASIGDTYGTALLFHMLVSTITLTLLAYQATKINSINVYAFSTIGYLCYTLGQVFHFCIFGNRLIEESSSVMEAA
YSCQRWDGSEEAKTFVQIVCQQCQKALTISGAKFFTVSLDLFASVLGAVVTYFMVLVQLK
Fig. 25. Putative recognition site of the anti-BmorOrco antibody. The amino acid sequence of MsexORCO (Grosse-Wilde et
al. 2011) is shown. The epitope highlighted in yellow completely matches the epitope, against which the anti-BmorORCO
antibody was raised. Identical amino acids of the epitope are shown in red.
MsexOR-1:
MIFMDDPLSKSIKDPRDYRYMKLFRSTLRLIGSWPGRDLKEEGATKYEIAPLYWVLVIKITCFVLTIIYLIENTNKLGFFEIGHVYITVFMTMI
TLSRSITLSLNPKYRRVMTKYITKMHLFYYKDMSDIALKTHIRVHKLSHFFTMYLSTQVVLGTVTFNIVPMYNNYKVGRFENNILVNDSYELSI
YFKTPTKFLSTLNGYIAITTFNWYSSYICSNFFCMFDLALSLLIFTVSGHFKILIHNLNNFPLPAVVSDSSKVLKTDEIQAPLYNKTEKKDITL
RLKQCIDYHREVLEFTQDISEAFGPMLFVYYLFHQVSGCLLLLECSQMDAAALMRYGLLTAVLFQQLIQLSVVVESVGTVTGYLKDAVYNVPWE
YMDTQDRKTVCIFLMNVQEPVHINALGLAKVGVQAMAGILKTSFSYFAFLRTVSN
MsexOR-4:
MKFFVDGSEIAHITKPQDIQYMQMLKFFTNSLAGWPIEAVEGIDGKKNFYWRNGLVVIAYAYFFGQVFYIYRYINDYTFLVMGHSYITVLMTIV
TIARHTLPYFKCYDDTTADFVHNIHLFNYRNKPGYYKEFHLKIHKISHAFSVYLCTLLVTGPSMFNGIPLYNNYASGAFSFNRSPNVTYEQAVS
LLLPFDDTNNFKGYFVVFLANCCVSYISSCCLCIYDLLLSLMVFHLWGHLKILTKTLDNFPKPGFLNPQAIEADPNKSLKFSDEELKVIHKKLG
ECVAHHQLISNFSTRMSNTFGLSLFIYYGFHQLSGCLLLLECAQLEAAAIICYGPLTLVVFQQLIQLSFIFELIGTVNEGLTDSVYCLPWEAMD
QGNKKIVFTFLRQSQKSMNLKALNMLSIGVQTMAKILKTTMTYFLMLQTIAKDES
Fig. 26. Putative recognition sites of the anti-BmorOR-3 antibody. The amino acid sequences of the pheromone receptor
candidates MsexOR-1 (top, accession number: FJ546086.1) and MsexOR-4 (bottom, accession number: HM595405.1) are
shown. Epitopes that are partly matched by the epitope used for the generation of the anti-BmorOR-3 antibody are
highlighted in yellow. Identical amino acids are marked in red, differing amino acids in blue.
70
2.5 Electrophysiology
2.5.1 Extracellular recordings from the isolated AME
Dissections were performed in extracellular ringer solution at different ZTs as described above
(2.3.3). Glass capillaries were pulled with a micropipette puller (P-97, Sutter Instrument Co.) and
manually broken to achieve a tip diameter around 200 µm for AME excision or somewhat smaller for
recording electrodes. For the latter the inside of the taper of the glass capillary was coated with wool
wax (adeps lanae anhydricus) to improve the seal between glass and tissue. After the capillary was
filled with extracellular Ringer solution the explanted AME tissue was partly sucked in, resulting in a
resistance between 0.1 and 1.5 MΩ. Electrical activity was amplified 1000 x using an extracellular
amplifier (EXT-01C/DPA 2F, NPI, Tamm, Germany) and measured as summed action potentials (SAPs)
in form of upward or downward deflections of the baseline as described before (Schneider and
Stengl 2005). Signals were digitized with a sampling frequency of 5 kHz using a Digidata 1322A
(Molecular Devices, Sunnyvale, CA, USA) and pClamp 8.2 software (Molecular Devices).
Recordings were performed in a tissue culture dish (diameter 3.5 cm, Greiner Bio One) containing
5 ml Ringer solution, which was perfused continuously with a flow rate of 10 ml/h at RT. Substances
were applied to the tissue in different doses by pressure ejection (Picospritzer II, General Valve
Corporation, Fairfield, NJ) via patch-clamp pipette-like glass capillaries (GC150T-10, Harvard
Apparatus Ltd., Edenbridge, UK): 2 pmol - 1.4 nmol 8-Br-cAMP (Sigma-Aldrich or Biolog, Bremen,
Germany), 90 - 310 pmol 8-pCPT-2'-O-Me-cAMP (007, Biolog), 4 - 400 pmol 8-Br-cGMP (SigmaAldrich or Biolog), 2 fmol - 1 nmol glutamate (Sigma-Aldrich), 75 fmol - 28 pmol PDF
(NSELINSLLSLPKNMNDAa, generously provided by Prof. Dick R. Nässel; NSEIINSLLGLPKVLNDAa,
purchased from Iris Biotech GmbH, Marktredwitz, Germany). The glass capillary for application as
well as the recording electrode was controlled via electronic micromanipulators (Luigs & Neumann
SM1-99630-S, Ratingen, Germany). Picrotoxin (PTX, Sigma-Aldrich) was applied via the perfusion
system at a concentration of 0.5 or 1.0 mM. Solutions applied via Picospritzer usually contained 5 %
food dye (McCormick and Company, Inc., Sparks, MD, USA) allowing for visual control of applications.
Fig. 27. Extracellular recording from an isolated accessory medulla. The
accessory medulla (AME) with associated neurons was isolated and partly
sucked into a suction electrode (SE), allowing for extracellular recording
of summed action potentials. Substances were applied into the vicinity of
the tissue with an application capillary (AC). Occasionally the tissue was
supported with another flame-sealed capillary (C). Scale bar: 300 µm.
Photo by Nils-Lasse Schneider.
71
2.5.2 Patch clamp recordings of AME cells
The patch clamp technique was developed by Bert Sakmann and Erwin Neher in the late 1970s and
early 1980s (Hamill et al. 1981). The technique was a powerful advancement of the 'voltage clamp',
which allowed the measurement of ionic currents through single ion channels. In 1991 the two
scientists were awarded with the Nobel Prize in physiology or medicine "for their discoveries
concerning the function of single ion channels in cells" (The Nobel foundation). To establish a patch
clamp recording a polished glass micro electrode with a tip diameter around 1-2 µm (= patch pipette)
is gently superimposed onto a cell. Negative pressure enables the formation of an intense connection
between the cell membrane and the glass of the patch pipette, resulting in a seal resistance in the
range of several GΩ, the so called 'gigaseal', which facilitates the sensitive measurements. Patch
clamp recordings might be performed in voltage clamp mode, where the cell is clamped to a certain
voltage and the ionic current necessary for compensation is measured, or in current clamp mode,
where the membrane potential of the cell is measured. Different recording configurations of the
patch clamp technique are illustrated in Fig. 28 (Hamill et al. 1981; Numberger and Draguhn 1996).
Fig. 28. Patch clamp configurations. A. The first
step towards a patch clamp recording is to bring
the patch pipette (profile shown in blue)
carefully in contact with the cell membrane. The
inner side of the membrane is shown in red and
the outer side in black. B. Gentle suction leads to
the formation of a tight connection between the
glass of the patch pipette and the cell membrane
in the range of several gigaohms (= gigaseal). In
the resulting cell-attached configuration single
channel openings in the respective membrane
patch can be measured. C. After formation of
the gigaseal further pulses of suction lead to a
disruption of the membrane patch and breaking
into the whole-cell configuration, where the
entirety of ion channels of the cell contribute to
the measured signal. D, F, G. Instead of breaking
into whole-cell mode it is also possible to retract
the patch pipette carefully (D). If the seal is
strong enough, the membrane outside of the
pipette will tear. F. Possibly the membrane will
re-seal, leading to formation of a small vesicle.
H. The vesicle might be destroyed by short air
exposure.
In
the
resulting
inside-out
configuration the cytosolic side of the
membrane faces the bath solution. E, G. After
breaking into the whole-cell configuration
carefully pulling back the patch pipette will tear
the membrane (E). G. Re-sealing of the
membrane results in the outside-out
configuration, where the cytosolic side of the
membrane faces the pipette. Figure modified
after (Hamill et al. 1981).
72
All patch clamp experiments in this thesis were performed in the whole-cell configuration (Fig. 29) on
AME-neurons, which were kept in culture for 1-14 days (days in vitro = DIV, 2.3.3). For experiments,
coverslips containing AME neurons were transferred from the tissue culture dish to a diamondshaped recording chamber (custom-made by the workshop of the University of Kassel), which was
connected to the perfusion system, allowing for laminar flow. Borosilicate glass capillaries (GC150TF7.5 or GC150F-7.5, Clark Electromedical Instruments) were used to fabricate patch pipettes with a tip
resistance of 2 - 8 MΩ. Usually patch pipettes were pulled and polished with a Zeitz DMZ-Puller (Zeitz
Instruments GmbH, Martinsried, Germany). Patch pipettes for the first experiments were pulled with
a P 87 or P 97 microelectrode puller (Sutter Instruments) and polished under optical control with a
custom-made microforge (workshop of the University of Regensburg).
Fig. 29. Steps to achieve a whole-cell recording. A,D,G. Illustrations of the patch pipette approaching the cell membrane
(A), the gigaseal formation (D), and rupture of the membrane patch (G). The cytosolic side of the membrane is shown in
red and the extracellular side in black. B, E, H. Current signals measured in response to a 5 mV-command potential pulse
(C). B. When the patch pipette is approaching the cell membrane, the pipette resistance increases and the current signal in
response to the test pulse flattens. E. During gigaseal formation, the current signal almost completely drops down, and
capacitive artifacts develop at the beginning and the end of the command potential pulse, which have to be compensated.
F. In the cell-attached configuration single channel openings can be measured. H. When the membrane patch is ruptured
(breaking into whole-cell mode), the steady state current in response to the test pulse increases, since the resistance of
the complete cell membrane is smaller than that of the small membrane patch. Simultaneously new capacitive artifacts
develop, which are larger and broader, since the capacity of the complete cell membrane is added to the circuit. I. After
compensation of capacitive artifacts and the series resistance, the current in response to voltage step protocols can be
measured.
73
Positioning of the patch pipette and seal formation was monitored with an inverted microscope
(Axiovert 135 TV, Zeiss, Jena, Germany) with phase contrast optics, using a correctable 40x objective
(LD ACHROPLAN 40x/0.60 Korr Ph2, Zeiss). Morphology of the cells was usually documented with a
Moticam 580 (Motic Deutschland GmbH, Wetzlar, Germany). Recordings were controlled with an
Axopatch 200B amplifier (Molecular Devices Corp., Union City, CA, USA). The signal was lowpass
Bessel-filtered at 5 or 10 kHz and digitized with a sampling rate of 20 kHz (Digidata 1322A and
pClamp 9 software, Molecular Devices Corp.).
Patch pipettes were filled with intracellular ringer solution and connected to the pipette holder. After
application of some positive pressure, the patch pipette was moved into the bath and positioned
near the target cell, before the offset potential was nullified. Patch pipettes as well as application
capillaries were positioned via electronic micromanipulators (Luigs & Neumann SM1-99630-S). After
seal formation the pipette capacitance was compensated. Then, the cells were clamped to -60 mV
and discrete pulses of suction were applied to rupture the membrane. After whole-cell configuration
was established the membrane potential of the cell was measured and whole-cell capacitance
transients were compensated. Series resistance compensation as well as liquid junction potential
correction was not performed. Leakage currents were corrected with the P/N leak subtraction
method, using two subsweeps, executed before the command waveforms. During the recording the
cell was stimulated with different voltage ramp or voltage step protocols to activate voltagedependent currents. Substances were applied via pipette or via pressure ejection using a
Picospritzer 2 (General Valve Corporation) or a pneumatic pico-pump (World Precision Instruments,
Sarasota, FL, USA). Additionally the perfusion system, driven by a peristaltic pump (Reglo Dig
MS/CA4-8C, Ismatec, IDEX Health & Science SA, Glattbrugg, Switzerland), was used to wash in or
wash out substances or different ringer solutions. The composition of all intracellular and
extracellular solutions is listed in the appendix (5.3.5).
2.6 Calcium Imaging
2.6.1 Calcium imaging experiments on heterologous expression
systems
The following chapter is a more detailed explanation of the calcium imaging experiments, which
were previously described in (Nolte et al. 2013): The majority of experiments was performed on
transiently transfected HEK 293 cells (2.3.6) and a few experiments were performed on transiently
transfected SF9 cells (2.3.7). All calcium imaging experiments on heterologous expression systems
were performed at the Max Planck Institute for Chemical Ecology (Jena) using fura-2 as calcium
indicator (Grynkiewicz et al. 1985). The membrane-permeable fura-2 acetoxymethyl ester (fura-2
AM, Molecular Probes, Invitrogen) was dissolved in DMSO at a concentration of 1 mM. Aliquots were
stored at -20 °C. The cells were loaded by adding fura-2 AM to the culture medium to achieve a final
concentration between 2.5 and 5 µmol/l. After an incubation for 30 - 60 min at RT the cells were
washed two times with ringer solution (for composition see 5.3.5) and incubated for at least further
15 minutes to allow for de-esterification of intracellular AM esters by unspecific esterases resulting in
74
a liberation and therefore activation of the calcium sensitive indicator. Cells grown on coverslips
were transferred into a bath chamber (RC-27, Warner Instruments, Hamden, CT, USA), which was
mounted with a P-6 platform (Warner Instruments) and a self made adaptor to the stage of an
epifluorescence microscope (Axioskop FS, Zeiss, Jena, Germany). The cells were monitored using a
40x objective (LUMPlanFI/IR 40x/0,80W, Olympus) or a 10x objective (ACHROPLAN 10x/0,30W Ph1,
Zeiss). Fura-2 was excited sequentially at wavelengths of 340 and 380 nm using a monochromator
(Polychrom V, Till Photonics, now FEI Munich GmbH, Germany) controlled by an imaging control unit
(ICU, Till Photonics) and Tillvision software (version 4.5, Till Photonics). Exposure times varied
depending on the objective used and the quality of the fura-2 loading to achieve good signal to noise
ratios for both excitation wavelengths. Emission for both excitation wavelengths was detected and
recorded at 510 nm using a PCO sensicam CCD camera (PCO, Kehlheim, Germany). Experiments had a
duration of minimum 5 minutes and the sampling interval was 5 s.
The
ORCO-agonist
VUAA1
(N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3yl)thio)acetamide, Jones et al. 2011) and the pheromone component (E,Z)-10,12-hexadecadienal
(E10,Z12-16:AL, bombykal) were synthesized by the Mass Spectrometry/Proteomics research group
at the Max Planck Institute for Chemical Ecology (Jena, Germany). Bombykal was synthesized via
oxidation of (E,Z)-10,12-hexadecadien-1-ol (E10,Z12-16:OH, bombykol). Bombykol was purchased
from Pherobank (Plant Research International, Wageningen, The Netherlands). (E,Z)-11,13pentadecadienal (E11,Z13-15:AL, C-15) was a kind gift from Prof. Dr. John G. Hildebrand (University
of Arizona, Tucson, USA). If not indicated otherwise chemicals were obtained from Sigma-Aldrich.
Stock solutions of the tested substances were prepared in ddH2O or in dimethyl sulfoxide (DMSO) if
not water-soluble. Working solutions were prepared by diluting stock solutions with ringer solution.
If dissolved in DMSO, the final content was 0.1 %. In addition to DMSO, bovine serum albumin (BSA)
was used to dissolve lipophilic substances. BSA was used in concentrations between 10-3 and 10-6 M.
In some experiments the bath chamber was perfused with a tubing pump (MS-CA, Ismatec, IDEX
Health & Science GmbH) to wash in or wash out substances. The diamond shaped bath chamber
allowed for laminar flow and therefore was better suited for perfusion than round cell culture dishes.
In other experiments stimuli were delivered via pipette or via rapid solution changer (RSC, Bio-Logic,
Claix, France) by gravity driven flow. For RSC applications a syringe rack was placed in the calcium
imaging setup containing reservoir syringes for different solutions tested. Syringes were connected
via polyethylene tubing with glass capillaries with an outer diameter of 1.7 mm (Hilgenberg GmbH,
Malsfeld, Germany). Glass capillaries were fixed with two O-rings to the RSC head, which provided
two guiding pins for straight positioning. The RSC head was mounted to a micromanipulator (Luigs &
Neumann) allowing for accurate positioning of the glass capillary and controlling from outside the
closed setup during experiments. Instead of equipping the RSC head with several glass capillaries for
application of different substances and using the automatic rotation function of the RSC head for
positioning, the head was equipped with only one capillary in the right position to ensure that only
this capillary touches the bath at a given time. For application a computer controlled valve had to be
opened to enable flow of the solution under test. To avoid optical artifacts, placing the capillary into
the bath, application and backing out the capillary each was performed between two measurements
(image acquisitions).
Because the majority of working solutions contained 0.1 % DMSO, control experiments were
performed to ensure that Ca2+ elevations after stimulation were not DMSO dependent. Therefore
75
either the complete experiment was performed in bath solution with a DMSO content of 0.1 % and
the cells were monitored without any further stimulation or the experiment was performed in
standard bath solution and 0.1 % DMSO solution was applied to the cells in the same way like other
solutions tested. Similar control experiments were performed for BSA.
To prevent pheromone contamination of the calcium imaging setup the air was evacuated with a
tube, which was connected to the air ventilation system. The end of the tube was directed to the
stage of the microscope. In addition the freely accessible surfaces of the setup were sequentially
cleaned with 70 % EtOH and 1 % Triton X-100 to remove possible pheromone contaminations.
2.6.2 Calcium imaging experiments on M. sexta ORNs
Calcium imaging experiments were performed on primary cell cultures of M. sexta ORNs (2.3.4) at
the University of Kassel, also using fura-2 AM as calcium indicator. Fura-2 AM was dissolved at a
concentration of 1 mM or 5 mM in DMSO and stored at -20 °C. For loading, the cells were incubated
in ringer solution (1 mM CaCl2, 5.3.5) containing 25 µM fura-2 AM for 120 min at RT. Other
conditions were also tested, but did not improve loading: Incubation times varied from 60 min to
240 min. The fura-2 AM concentration was varied from 10 µM to 50 µM. Occasionally, 20 % Pluronic
F-127 (Invitrogen), a macromolecular surfactant (Cohen et al. 1974), was used to dissolve fura-2 AM,
or a volume of 5 - 10 µl of Pluronic F-127 was added to 1 ml of loading solution. In some experiments
blockers for multidrug resistance transporters were present in the loading solution. Probenecid
(Invitrogen) was dissolved in HBSS at a concentration of 250 mM, used at a final concentration of 2.5
mM, and stored at -20 °C. Verapamil (Sigma-Aldrich) was dissolved in ddH2O at a concentration of
100 mM, used at a final concentration of 500 µM, and stored at 4 °C. After loading the cells were
washed two times with ringer solution and incubated for at least another 30 min, allowing for
complete de-esterification of intracellular fura-2 AM.
For the experiments the coverslip containing the cells was transferred into a recording chamber,
which was identical to the one, used for patch clamp experiments. The bath chamber was mounted
to an upright microscope (Examiner D1, Zeiss) and connected to the perfusion system. The cells were
monitored with an 20x water immersion objective (N-Achroplan 20x/1.0W, Zeiss). The calcium
indicator was excited sequentially at 340 nm and 380 nm using a Polychrom 5000 monochromator
(Till Photonics) and an ICU (Till Photonics). Exposure times were adjusted to achieve good signal to
noise ratios for both excitation wavelengths, with the exposure time for 340 nm always being higher
than the exposure time for 380 nm. Emission was detected at 510 nm using an Andor 885 CCD
camera (Andor, part of Oxford Instruments, Belfast, UK).
The cells were stimulated with different ORCO agonists and antagonist (Fig. 30). VUAA1 and VUAA4
were synthesized by the Mass Spectrometry/Proteomics research group at the Max Planck Institute
for Chemical Ecology (Jena, Germany). ORCO ligand candidates (OLC12 and OLC15) were kindly
provided by Dr. Charles W. Luetje (Miller School of Medicine, University of Miami), and the amiloride
derivatives 5-(N,N-hexamethylene)amiloride (HMA) and 5-(N-methyl-N-isobutyl)amiloride (MIA)
were purchased from Sigma-Aldrich. All substances tested were dissolved in DMSO and stored at 20 °C. For the experiments the stock solutions were diluted in ringer solution (6 mM CaCl2, 5.3.5)
76
resulting in a DMSO content of 0.1 %. Stimuli were delivered via pipette application or via the
perfusion system, consisting of one peristaltic pump (2132 Microperpex, LKB, Stockholm, Sweden)
for continuous perfusion of the bath chamber with ringer solution and another peristaltic pump for
application of the substances tested.
Fig. 30. Structures of different ORCO agonists and antagonists. Substances of the VUAA1-family such as VUAA4, OLC12,
and OLC15 only differ in minor chemical modifications of the VUAA1 structure and seem to be specific ORCO agonists
(VUAA1, VUAA4, and OLC2) or antagonists (OLC15). Amiloride derivatives such as 5-(N,N-hexamethylene)amiloride (HMA)
and 5-(N-methyl-N-isobutyl)amiloride (MIA) deviate by a greater amount from the VUAA1 structure and are less specific
ORCO antagonists, which are reported to block a variety of ion channels.
2.7 Data analysis
2.7.1 Immunocytochemical analysis of heterologous M. sexta ORCO
expression
After scanning representative areas of the specimens, confocal stacks in the Leica image file format
(.lif) were imported to Fiji/ImageJ software (version 1.49, National Institutes of Health, Bethesda,
MD, USA) for further analysis using the Bio-Formats plugin (Laboratory for Optical and Computational
Instrumentation, Madison, WI, USA). Heterologous M. sexta ORCO expression was qualitatively
analyzed, i.e. it was solely examined whether ORCO-like immunoreactivity could be detected,
whereby the strength of the signal was not evaluated. First maximum projections were computed,
then cells were counted, and the percentage of ORCO-expressing cells was calculated.
The subcellular localization of heterologously expressed ORCO was only analyzed for a few cells on a
random basis. To illustrate the subcellular localization optical sections of the middle range of the cells
(in z-direction) were used for fluorescence intensity analyses, whereby the intensities for different
77
fluorescence signals along a line were compared, and for the generation of three-dimensional
surface plots, whereby the pixel intensities of an area were three-dimensionally plotted.
2.7.2 Analysis of extracellularly recorded electrical activity of AME
neurons
Electrical activity of the isolated AME neurons was extracellularly recorded using pClamp 8.2
software and imported to Spike2 software (version 7, Cambridge Electronic Design, Cambridge, UK)
for further analysis. To evaluate the firing pattern of the recorded neurons and the effects of drug
application on the electrical activity, a threshold-based event detection was performed (Fig. 31). The
timing of the events was used to calculate the mean frequency and interevent-intervals.
Fig. 31. Threshold-based event detection. A. Recording
trace showing summed action potentials (SAPs) as upward
deflections from the baseline. The threshold (dotted line) is
set at a position, ensuring that it is reached by the spikes of
interest, but not by the noise. B. The vertical lines
symbolize events. Each time the threshold is crossed by an
SAP (A), an event is generated in the event channel.
The interevent-intervals were used to prepare interevent-interval histograms (Fig. 32), which were
computed with bin sizes between 0.5 and 2.0 ms. A distinct peak in the interevent-interval histogram
indicates regular firing of the neurons with the same interevent-interval, i.e. firing with the same
ultradian period (Schneider and Stengl 2005). Furthermore the coefficient of variation (CV; equals
the standard deviation divided by the mean) was calculated for the interevent-intervals, using
GraphPad Prism (version 5, GraphPad Software, La Jolla, CA, USA). The CV is a measure for the
degree of variability in relation to the mean of the population and can be used to estimate regular
firing patterns. According to different publications regular firing is indicated by interevent-interval CV
values < 0.5 or ≤ 0.35 (Young et al. 1988; Prut and Perlmutter 2003).
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Fig. 32. Calculation of an interevent-interval
histogram. A. The vertical dashes symbolize events,
each of which was detected, when the recorded
electrical activity exceeded the threshold (Fig. 31).
Each number between two events indicates the size
of the respective interevent-interval in seconds.
B. Intervals of equal length (defined by the chosen
bin-size) are counted and the number is represented
as bar (or bin) in the interevent-interval histogram.
Here, all intervals are shown as an integer number of
seconds and the bin size was set to 1 s. Since intervals
of 2 s occur more frequently than other intervals, the
histogram shows a peak at 2 s. In a more realistic case
the intervals would not be restricted to integer
numbers and rather be in the range of some
milliseconds. However, with a bin-size of 1 s all
intervals equal or bigger than 1.0 s and smaller than
2.0 s would be included in the 1 s-bin of the
histogram.
Fig. 33. The instantaneous frequency plot. A. The vertical dashes symbolize events, originating from threshold exceeding
summed action potentials generated by three assemblies of simultaneously spiking cells (black, blue, and red). Each
assembly fires with a frequency of 6.25 Hz. Within one assembly the cells spike with the same phase, but cells of different
assemblies spike with a constant phase difference. Cells of the second assembly (blue) spike 20 ms later than cells of the
first assembly (black). The third assembly (red) spikes 40 ms later than the second one (blue). Short inhibition of the third
assembly (red symbol) results in an increase of the blue-red interevent-interval from 40 ms to 60 ms and a decrease of the
red-black interevent-interval from 100 ms to 80 ms. B. Each point in the instantaneous frequency plot is the reciprocal
value of the interevent-interval between the event at that time and the preceding one. In the beginning three recurring
interevent-intervals of 20 ms (black-blue), 40 ms (blue-red), and 100 ms (red-black) result in three instantaneous
frequency bands at 50 Hz (blue, 1/20 ms), 25 Hz (red, 1/40 ms), and 10 Hz (black, 1/100 ms). A band in the instantaneous
frequency plot reflects a stable, recurring interevent-interval and thus, indirectly an assembly of cells. If one of the three
assemblies is phase-shifted (in this case: delayed), the effect will hit two of three interevent-intervals and thus
instantaneous frequency bands. Here, short inhibition of one assembly leads to a decrease of one band from 25 Hz to
16.7 Hz (red, 1/60 ms) and an increase of one band from 10 Hz to 12.5 Hz (black, 1/80 ms). However, the third intereventinterval and the resulting instantaneous frequency band at 50 Hz is not affected.
Additionally the interevent-intervals were used to compute the instantaneous frequency, which is
defined as the reciprocal value of the interevent-intervals. The reciprocal value of each interevent79
interval is illustrated as one point in the instantaneous frequency plot (Fig. 33). When all cells
regularly fire with the same interevent-interval (i.e. with the same or integer multiples of the same
frequency) and with the same phase, a narrow band of points will appear in the instantaneous
frequency plot. If different assemblies of cells are recorded, within which the cells fire with the same
frequency and phase, and between which the cells fire with the same frequency but a constant phase
difference, multiple stable interevent-intervals will occur, and thus multiple, parallel instantaneous
frequency bands (Schneider and Stengl 2005). Since a phase-shift of one assembly always affects two
interevent-intervals (Fig. 33) it is difficult to draw conclusions about changes of instantaneous
frequency bands. In contrast, irregular firing results in a broad cloud of points in the instantaneous
frequency plot, without any defined bands. In the case of different assemblies, firing with different,
non-integer frequencies and different phases would lead to crossing instantaneous frequency bands
(Pikovsky et al. 2001; Schneider and Stengl 2005).
Fig. 34. Construction of an auto-correlogram. A. Event channel with vertical lines symbolizing events. Each event serves as
a trigger for one sweep of analysis, in which the times of the events, occurring in a selectable range (here: 0.2 s), are
detected and included in a histogram. Four sweeps of analysis, triggered by the first four events, are illustrated. B. For
each sweep of analysis, a histogram is generated, showing the number of events at the time of occurrence of the events,
which logically equals one. The correlation of each event with itself at time 0.00 s is ignored. Finally, the single histograms
are summed to obtain an auto-correlogram. C. Auto-correlogram including the first four sweeps of analysis. D. The autocorrelogram, generated for 10 min of activity, shows six evenly distributed, defined peaks, indicating regular spiking
activity with the same interevent-interval.
Eventually the timing of the events was used to perform auto-correlations to visualize regular firing
patterns. While in a cross-correlation one event channel is correlated with another event channel
(the trigger channel), in an auto-correlation the event channel is correlated with itself. It produces a
measure of the likelihood of an event occurring at a certain time after another event (Fig. 34).
80
Therefore, peaks in an auto-correlogram reflect times of increased discharge probability. If the
recorded cells fire regularly with the same interevent-interval, multiple, equally spaced peaks will
occur in the auto-correlogram (Tepper et al. 1995; Schneider and Stengl 2005). Here, autocorrelations were performed using a bin width of 0.5 - 2.0 ms over 5 - 30 min of neuronal activity.
The activity was classified as regular, if four or more peaks were detected in the respective autocorrelograms.
2.7.3 Analysis of whole-cell patch clamp recordings of single AME
neurons
Whole-cell currents of single AME neurons, which were recorded in response to voltage ramp or
voltage step protocols, were analyzed with pClamp software (version 10.4, Molecular Devices Corp.).
Current-voltage (I-V) relationships were calculated at different positions of the current responses to
analyze different components of the whole-cell currents, such as transient (= inactivating), sustained
(= non-inactivating), or tail currents. By means of the I-V curves the frequency of the current
components, reversal potentials (Vrev), as well as activation, inactivation, and peak voltages were
determined. To evaluate the development of certain current components during a recording the time
course of the peak currents or the time course of the area under the I-V curves (AUC) was analyzed
with respect to spontaneous or drug-dependent changes. Since pClamp software did not feature the
construction of AUC time courses, this analysis was performed using Matlab software (version
R2012a, The MathWorks Inc., Natick, MA, USA; script by Dr. Achim Werckenthin). For this purpose
the respective current response data and the corresponding time within the recording were
exported. Eventually the Matlab script was used to access the data and plot the respective current
responses, I-V curves, as well as AUC time courses.
Spikes measured in patch clamp recordings were analyzed with pClamp software. The spike
amplitude was defined as the distance between the peak potential and the most negative potential
of the following hyperpolarization. The spike duration was measured at half-maximal amplitude
(Fig. 35).
Fig. 35. Spike duration. The duration of spikes was defined
as the distance between the points, at which the
membrane potential crossed the half-maximal amplitude in
depolarizing direction and hyperpolarizing direction.
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2.7.4 Analysis of calcium imaging data of heterologous expression
systems
The following section was published previously with almost identical wording (Nolte et al. 2013).
Tillvision software was used to subtract background fluorescence and to calculate the ratio of
fluorescence data resulting from excitation at 340 nm and 380 nm (F340/F380, Fig. 37). The ratio was
calculated using the following formula:
The scale factor was required since images were kept in integer type buffers. It was set to 1000 by
default. Both differences of the formula were clipped to zero, if the value was smaller than 30. A
decrease in the ratio indicated a decrease in the Ca2+ concentration and an increase indicated an
increase respectively (Grynkiewicz et al. 1985). Regions of interest (ROIs) were defined by marking
the profile of each cell (Fig. 36). If the number of cells was far higher than 100, the number of ROIs
was limited to 100. For each ROI the average ratio was calculated and the values for all ROIs over the
course of time of an experiment were exported as text file. All text files and an excel file containing
required metadata were accessed by Matlab software (script by Achim Werckenthin, University of
Kassel) for further analysis. First the data (i.e. F340/F380-ratios for each cell) were normalized by
calculating the mean of the first ten values of a measurement and the percentage deviation from the
mean for each value (Fig. 37). Then the percentage of responding cells for each experiment was
determined. Cells were considered as responding cells, if the percentage deviation exceeded the 20fold standard deviation of the values measured before application or of the first ten values, if cells
were monitored without stimulus application. The cells were differentiated and grouped according to
critical characteristics. For example, ORCO positive cells (i. e. transfected with MsexOrco and
optionally cotransfected with a pheromone receptor candidate (MsexOr-1 or MsexOr-4) and/or
MsexSnmp-1) were differentiated from ORCO negative cells (optionally transfected with MsexOr-1,
MsexOr-4 and/or MsexSnmp-1, but not with MsexOrco).
Fig. 36. Definition of ROIs. The shown HEK 293 cells
were loaded with the calcium indicator fura-2 AM.
After excitation at 380 nm the fluorescence was
detected at 510 nm. Low fluorescence intensity is
indicated by dark blue, high fluorescence intensity
by red. For further analysis regions of interest (ROIs)
were defined. ROI 0 marks the background, while
ROI 1-26 mark the outlines of the HEK 293 cells.
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Fig. 37. Presentation of fura-2-based calcium
imaging data. A-D. Calcium imaging data for one
HEK 293 cell, which was loaded with the calcium
sensitive dye fura-2 AM. The ratiometric calcium
indicator was excited sequentially at 340 and 380
nm and the resulting fluorescence was detected at
510 nm. A. Kinetic of the fluorescent intensity
resulting from excitation at 340 nm (F340), showing
an increase. B. Kinetic of the fluorescence intensity
resulting from excitation at 380 nm (F380), showing
a decrease. C. Ratio of the fluorescence intensities
resulting from 340 and 380 nm (F340/F380), showing
an increase. The increase of F340, the decrease of
F380, and the resulting increase of the ratio indicates
an increase of the intracellular calcium
concentration. D. For further analysis the F340/F380
data were normalized by presenting each value as
percentage deviation from the mean of the first ten
values of the data set (% Δ(F340/F380)).
2.7.5 Analysis of M. sexta ORN calcium imaging data
Definition of ROIs, calculation of the fluorescence ratio (F340/F380), and data export as text files was
performed with Tillvision software as described before (2.7.4). ROIs were limited to presumptive
ORNs in the heterogeneous primary cell cultures (2.3.4). Further analysis was performed with Matlab
software using a script written by Andreas Nolte (University of Kassel). The Matlab script was also
used to determine the percentage of responding cells and to plot the kinetics of the fluorescence
ratio of all cells. However, it differed in some aspects from the script used for the analysis of the
heterologous expression data (2.7.4): No metadata were accessed, i.e. the stimulation frame was
entered manually for each run. The search for responding cells was performed with absolute values
of the fluorescence ratio. If the fluorescence ratio exceeded a certain standard deviation from the
mean of all values before application, the corresponding cell was classified as active cell. Here, the
five-fold standard deviation was chosen. Additionally, the scripts allowed to search for inhibited cells,
defined as cells with fluorescence ratios falling below a certain standard deviation from the mean of
the values before application. A normalization of the data was performed only for plotting. For this
purpose each value was represented as percentage deviation from the mean of the values measured
before application.
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2.7.6 Statistical analysis
All statistical tests were performed with Prism software (version 5.01, GraphPad Software, La Jolla,
CA, USA). The D'Agostino-Pearson omnibus test (K2) was used to test for normality. The appropriate
statistical test to compare data groups was chosen depending on a possible Gaussian distribution
(parametric or non-parametric test), on the number of groups, and on a possible pairing of the data.
For all statistical tests a significance level of α=0.05 was chosen. Data were represented as box plots
consisting of upper and lower quartile with median and whiskers from minimum to maximum. If the
sample size was very small (n < 5), data were represented as scatter plots, with each symbol
representing one sample.
2.8 Preparation of figures
Preliminary figures were created with Matlab (version R2012a, The MathWorks Inc., Natick, MA,
USA), Prism (version 5.01, GraphPad Software, La Jolla, CA, USA), or Origin software (version 9.0.0G,
OriginLab Corporation, Northampton, MA, USA). Chemical structures were drawn with ChemSketch
(version 14.01, Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). Images were
edited using IrfanView (freeware by Irfan Skiljan, Vienna University of Technology, Vienna, Austria) or
Fiji/ImageJ software (version 1.49, National Institutes of Health, Bethesda, MD, USA. Final editing of
all figures was performed with Corel Draw X3 software (Corel Corporation, Ottawa, Ontario, Canada).
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Results
3 Results
3.1 Electrophysiological characterization of the R. maderae
central clock network
3.1.1 Network activity of isolated AME neurons
In this part of the thesis the central pacemaker neurons of R. maderae, the AME neurons, were
analyzed at the network level. Therefore, the AME with surrounding somata was excised (Petri and
Stengl 1999; Reischig and Stengl 2003a) and used for extracellular recordings with a suction
electrode (Funk 2005; Schneider and Stengl 2005, 2006, 2007). This technique allowed for long-term
recordings of summed action potentials (SAPs), generated by several cells of the tissue (Schneider
and Stengl 2005), lasting for several days (Funk 2005; Schneider and Stengl 2007). A total of 126
recordings (126 AMAE from 120 Madeira cockroaches) were performed, including 79 recordings from
previous experiments (Funk 2005). The data were re-analyzed in a broader context. Parts of my
conclusions were drawn before (Funk 2005) and published, respectively (Schneider and Stengl 2005).
The dissections of AMAE were performed throughout the day, not at a particular Zeitgeber time (ZT).
However, more than half of the dissections occurred between ZT 4 and ZT 9 (64 of 126 dissections)
and more than one third between ZT 17 and ZT 22 (36 of 126 dissections, Fig. 38). Depending on the
exact position of the active cells relative to the bath solution (the indifferent electrode respectively)
and the electrode solution (the recording electrode respectively) the SAPs appeared as positive or
negative deflections from the baseline with amplitudes up to 400 µV. Since the experiments did not
aim at an investigation of potential circadian rhythms in the electrical activity, dissections as well as
experiments were performed under illumination, irrespective of the animal's physiological (temporal)
state. Therefore, no time-dependent firing patterns were analyzed.
Fig. 38. Dissection times. The figure shows the temporal
distribution of dissections of AMAE, used for extracellular
recordings. Dissections cumulated around Zeitgeber time
(ZT) 7 and ZT 19.
First, the firing pattern of the recorded neurons was analyzed. In 63.1 % of the analyzed recordings
(n = 77 of 122) the coefficient of variation (CV) of the interevent-intervals was either smaller or equal
0.35 (Young et al. 1988) or multiple defined peaks (≥ 4 peaks) were detected in the respective
autocorrelograms (Tepper et al. 1995; Schneider and Stengl 2005), indicating highly regular firing. In
these recordings the cells (or assemblies of cells) fired with the same or integer multiples of the same
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Results
ultradian period (i.e. interevent-interval) without phase difference, resulting in one peak in the
interevent-interval histogram and correspondingly in a narrow band in the instantaneous frequency
(1/interevent-intervals) plot (Schneider and Stengl 2005, Fig. 39).
Fig. 39. Neurons of the isolated AME fire regularly. A-E. Extracellular recording from an isolated AME. A. Recording trace
of 10 s duration, showing regularly occurring summed action potentials (SAPs) with different amplitudes as downward
deflections from the baseline. B. The mean frequency of the SAPs stably fluctuated around 25 Hz. C, D. The instantaneous
frequency (1/interevent-intervals) plot (C) shows a narrow band around 21 Hz corresponding to the peak of the intereventinterval histogram (D) at 47.5 ms. E. The autocorrelogram shows multiple defined peaks caused by regular intereventintervals, indicating regular spiking activity.
Another characteristic firing pattern of the AME network was the spontaneous alternation of highactivity phases with low-activity phases, hereinafter referred to as oscillations of the neuronal
network activity or bursting. Spontaneous oscillations or bursting could be observed in 47.2 % of the
analyzed recordings (n = 50 of 106). The oscillation pattern within these recordings was highly
variable, including recordings with phases of absolute quiescence (Fig. 40) and others with phases of
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Results
lower activity between the phases of rapid firing. In some recordings oscillations occurred for several
hours, in others only for some minutes, before the activity switched back to a constant firing mode
(Fig. 40) or disappeared. The duration of the high- and low-activity phases varied from short bursts of
a few SAPs with interburst-intervals of a few hundred milliseconds (Fig. 121) to minute-long
durations (not shown). Underlying mechanisms of the oscillations as well as potential regularities or
periodicities were not analyzed. However, in two recordings very regular interburst-intervals were
observed, which resulted in a narrow band in the respective instantaneous frequency plots
(Fig. 43, Fig. 121).
As described by Schneider and Stengl (2005), the AME network is characterized by widespread
inhibitory synaptic interactions, which play a role in synchronization or rather phase locking of
neuronal assemblies. When Ca2+-free ringer solution was applied to inhibit neurotransmitter release
and thus chemical synaptic transmission, the network activity of the recorded neurons increased in
97.9 % of the recordings (n = 46 of 47, Fig. 41), indicating a disinhibition of previously inhibited
neurons and confirming previous results (Schneider and Stengl 2005). In a single recording
(n = 1 of 47) the activity was not changed by Ca2+-free solution (not shown). In 52.2 % of those
disinhibitions (n = 24 of 46) the neurons still fired regularly with a similar interevent-interval resulting
in a band in the instantaneous frequency plot. In one recording, where the neurons first fired
regularly with the same interevent-interval before the activity spontaneously ceased, blocking of
synaptic transmission disinhibited the neurons, which now fired regularly with two recurring
interevent-intervals, resulting in two instantaneous frequency bands (Fig. 41) as previously reported
(Schneider and Stengl 2005).
Next to the disinhibitory effect, burst firing and oscillations of the electrical activity were also
enhanced in the absence of synaptic transmission. This was the case in 76.6 % of the respective
recordings (n = 36 of 47, Fig. 43). In 10.6 % of the recordings (n = 5 of 47) the disinhibitory effect of
the Ca2+-free solution was only transient, and subsequently the activity was reversibly inhibited
(Fig. 42), suggesting that the AME network also contains activating chemical synapses, although to a
lesser extent than inhibitory synapses.
Interestingly, it could be observed that traces of Ca2+ were sufficient, to trigger minute-long activity
changes during the inhibition of synaptic transmission with Ca2+-free solution: When a few nanoliters
of the standard saline used for extracellular recordings (containing 6 mM CaCl2) were applied to the
vicinity of the AME-tissue, the activity pattern was rigorously changed (Fig. 43). Therefore, it was of
great importance to ensure that all substances were always diluted in the appropriate ringer
solution.
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Results
Fig. 40. Neurons of the excised AME show spontaneous bursting and stimulus-independent activity changes. A-E.
Extracellular recording from an excised AME. A. Original recording trace, showing summed action potentials (SAPs) as
upward deflections from the baseline. The arrows (d and e) mark the positions of the details, which are magnified in D and
E. The activity pattern of the neurons spontaneously switched from a bursting mode to a constant firing mode,
accompanied by a reduction of the SAP-amplitudes (compare D and E). B, C. The corresponding mean frequency (B) and
instantaneous frequency (1/interevent-intervals) plots (C) also illustrate the oscillatory activity and the activity change,
which was combined with a strong frequency rise, followed by a decrease to a new stable level. C. The formation of a
broad instantaneous frequency band indicates constant, more regular firing with similar interevent-intervals, as can also
be seen in E.
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Results
Fig. 41. In the absence of inhibitory synaptic interactions AME neurons still fire synchronously. A-E. Extracellular
recording from an isolated AME. A. Original recording trace over a duration of 900 s, showing summed action potentials
(SAPs) as upward and downward deflections from the baseline. Positive SAPs were inhibited in the beginning and
2+
disinhibited only after washing in low Ca ringer solution (indicated by the grey bar) to block synaptic transmission. The
arrow marks the position of the detail that is magnified in D. B. The increase of the mean frequency illustrates the
disinhibition. C. The instantaneous frequency (1/interevent-interval) plot shows the development of a new stable
condition in the absence of chemical synaptic transmission. Two instantaneous frequency-bands around 65 Hz and 40 Hz
indicate two recurring interevent-intervals, which can also be seen in the detail of the recording trace (D) and the
interevent-interval-histogram (E, generated over the last 300 s of the shown recording trace).
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Fig. 42. The AME network also contains activating chemical synapses. A-C. Extracellular recording from an isolated AME.
A. Original recordings trace with a duration of 30 min, showing summed action potentials as negative deflections from the
2+
baseline. Blocking synaptic interactions with low Ca -ringer solution (grey bar) reversibly inhibited the electrical activity.
B, C. Mean frequency (B) and instantaneous frequency (C, 1/interevent-intervals) illustrate a short activation and the
2+
subsequent almost complete inhibition after deprivation of Ca . After recovery the instantaneous frequency band
broadened (C), indicating a loss of synchrony.
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2+
Fig. 43. Small changes in the extracellular Ca -concentration are sufficient to induce changes in the firing pattern. A-E.
2+
Extracellular recording from an isolated AME in low Ca ringer solution. A. Original recording trace showing summed
action potentials (SAPs) as negative and positive deflections from the baseline. The arrowhead marks the application of
2+
ringer solution containing 6 mM CaCl2 (10 nl via Picospritzer). Application of Ca led to a transient increase of the positive
2+
SAPs' amplitude, while the negative SAPs were not affected. B. The mean frequency of the positive SAPs decreased Ca dependently. C. The instantaneous frequency (1/interevent-intervals) plot shows a broad band in the absence of
2+
2+
extracellular Ca and thus synaptic transmission. Ca application resulted in the transient broadening of the band and the
appearance of a narrow band at a lower frequency (10 Hz) as well as several points at a higher frequency, indicating the
appearance of several different interevent-intervals. D, E. Details of the recording trace (marked by the arrows d and e in
2+
A). The temporary presence of extracellular Ca switched the neurons to a bursting mode, resulting in small intereventintervals (during the bursts) as well as long interevent-intervals (between the bursts, corresponding to the instantaneous
frequency band around 10 Hz).
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Results
3.1.2 Glutamate inhibits AME neurons
Schneider and Stengl (2005) demonstrated that the Cl- channel antagonist PTX simulated the effect
of Ca2+-free saline, and that the neurotransmitter GABA effectively inhibited AME neurons, indicating
a major role of GABAergic synaptic interactions in the AME. However, the contribution of other
inhibitory neurotransmitters acting via Cl- channels was not examined. Since glutamate was reported
to signal via glutamate-gated Cl- channels (GluCl) in D. melanogaster (Cully et al. 1996), a potential
contribution of this neurotransmitter was investigated. The effect of PTX was confirmed: When PTX
was washed into the bath at a concentration of 0.5 or 1 mM, the AME network activity was
disinhibited in all preparations (n = 8, Fig. 44). Glutamate was applied at different concentrations via
pressure ejection using a Picospritzer (n = 4 recordings). The respective dissections were performed
at different ZTs (ZT 10, 20, 21, and 23). Glutamate dose-dependently inhibited the AME network
activity: Doses of 2 fmol - 10 pmol glutamate did not change the electrical activity (n = 4 recordings),
while doses of 20 pmol - 1 nmol inhibited the firing rate to a different extent (n = 2 of 3 recordings,
Fig. 45). Inhibitions eventuated at different ZTs (ZT 10, 11, 22, and 23) with dose-independent delays
of 1.4 s - 30 s. In contrast, the duration of the inhibition was dose-dependent and ranged from 36 s
(20 pmol) to 409 s (1 nmol). Inhibitions in Ca2+-free saline indicated direct effects (n = 1 recording,
Fig. 45). Thus, glutamatergic synapses appeared to contribute to the widespread inhibitory synaptic
interactions of the AME network and to be involved in PTX-dependent disinhibitions. To test,
whether glutamate-dependent inhibitions were mediated via PTX-sensitive Cl- channels, glutamate
was applied, while the AME network activity was PTX-dependently disinhibited (n = 1 recording,
0.5 mM PTX). Remarkably, glutamate still dose-dependently inhibited the activity (Fig. 46).
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Fig. 44. PTX disinhibits AME neurons. A-E. Extracellular recording of an isolated AME. A. Original recording trace showing
summed action potentials (SAPs) as upward deflections from the baseline. The grey bar marks the application of the
Cl channel antagonist picrotoxin (PTX, 0.5 mM) via the perfusion system, resulting in an increase of the SAP frequency (see
also B). The small bar (d) marks the position of the detail shown in D. C. The instantaneous frequency (1/intereventintervals) plot shows the PTX-dependent formation of a narrow, increasing band, indicating that the neurons still fired
synchronously in the presence of PTX. D, E. Synchronous firing with the same interevent-interval is also illustrated in the
detail of the recording trace (D) and the interevent-interval histogram (E) showing a peak around 125 ms.
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Results
Fig. 45. Glutamate dose-dependently inhibits AME neurons. A-F. Original recording traces of an extracellular recording of
2+
an isolated AME. Ca -free ringer solution was used to block synaptic transmission. The arrows above the recording traces
-14
-11
indicate the application of different doses of glutamate (via Picospritzer). While application of 10 - 10 mol glutamate
-10
-9
did not affect the electrical activity (A-D), application of 10 mol resulted in a mild inhibition (E) and 10 mol in a strong
inhibition (F).
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Results
Fig. 46. Glutamate inhibits AME neurons in the presence of PTX. A-C. Extracellular recording of an isolated AME,
performed in the presence of the Cl channel antagonist picrotoxin (PTX, 0.5 mM). The arrows above the recording trace
(A) mark the applications of glutamate (via Picospritzer). Despite the presence of PTX dose-dependent inhibitions of
glutamate could be observed in the recording trace (A) and the corresponding mean frequency (B) or instantaneous
frequency (1/interevent-intervals) plot (C). The narrow instantaneous frequency band indicates synchronous firing with
the same interevent-interval (C).
3.1.3 Different effects of PDF on neurons of the isolated AME
Next, the effects of the neuropeptide PDF on the AME network were characterized further.
Schneider and Stengl (2005) reported PDF-dependent, transient inhibitions and activations of the
neuronal activity. Since inhibitions but not activations persisted in the absence of synaptic
transmission, inhibitions were suggested to be direct effects and activations to be indirect effects
(disinhibitions, Schneider and Stengl 2005). Here, PDF was applied at different doses (75 fmol 28 pmol) in 58 recordings, of which 37 recordings were analyzed. The dissections of the AMAE as well
the PDF applications were performed throughout the day, but dissections cumulated between ZT 4
and ZT 11 and applications between ZT 6 and ZT 13 (Fig. 120). In 51.4 % of the analyzed recordings
performed in standard extracellular saline (n = 18 of 35) PDF did not affect the AME network activity
(Tab. 7, Fig. 54). In the remaining recordings different effects were observed: In 5.7 % of the
recordings (n = 2 of 35) PDF increased the firing rate (Fig. 47, Fig. 122) and in 34.3 % of the recordings
(n = 12 of 35) it decreased the firing rate (Fig. 48, Fig. 54, Fig. 122). Furthermore, PDF increased
bursting behavior or caused oscillations in the firing rate in 20.0 % of the recordings (n = 7 of 35,
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Results
Fig. 48, Fig. 49, Fig. 54, Fig. 122, Tab. 7). In some recordings (n = 3) the application of PDF had
multiple effects: In one recording, for instance, one population of cells first started to oscillate and
then was inhibited, while another population of cells first increased spiking activity and started
bursting, before its activity completely disappeared after several minutes (Fig. 122). In another
recording, PDF caused oscillations and an inhibition of the network activity and simultaneously
synchronized the recorded neurons, resulting in the formation of a narrow instantaneous frequency
band (Fig. 48). When PDF was applied in the absence of synaptic transmission (in Ca2+-free solution,
n = 2 recordings) or in the presence of PTX (n = 1 recording), no changes in the electrical activity were
observed (not shown). PDF-dependent changes of the electrical activity had different delays (< 1 s to
26 min) and durations (18 s to > 2 h), which apparently were not dose-dependent. Thus, PDF caused
transient as well as long-lasting activity changes. In some cases the recording was terminated after a
longer period of quiescence or oscillations, making it impossible to evaluate the exact duration of
long-lasting effects. Since PDF applications cumulated between ZT 6 and ZT 13, the PDF effects also
cumulated in this time slot (not shown), and no time-dependency for different PDF effects was
detected.
Fig. 47. PDF transiently activates AME neurons. A, B. Recording traces of an extracellular recording from an isolated AME
showing summed action potentials (SAPs). Application of PDF (2 nl, 150 fmol, via Picospritzer) transiently activated the
recorded neurons (A), while a control application (2 nl) had no effect (B). The data were re-analyzed from (Funk 2005).
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Results
Fig. 48. PDF inhibits and synchronizes AME neurons. A-C. Extracellular recording from an isolated AME. A. Original
recording trace showing summed action potentials (SAPs) as negative deflections from the baseline. The arrows mark the
application of PDF (left: 1.35 pmol, right: 1.5 pmol, via Picospritzer) and the arrowheads (d and e) mark the positions of the
details, magnified in D and E. After PDF application first the large-amplitude SAPs failed to appear and in the end of the
recording trace also small-amplitude SAPs. B. The mean frequency illustrates the PDF-dependent inhibition and shows a
period of oscillations starting 200 s after the second PDF application. C. The instantaneous frequency (1/intereventintervals) plot indicates that the PDF-dependent inhibition and oscillations were accompanied by a synchronization: After
PDF application a narrow instantaneous frequency band developed, indicating firing with similar interevent-intervals.
Before the electrical activity was completely blocked (23 minutes after the second PDF application), the band broadened
again. D, E. Details of the recording trace, showing more activity and noise before PDF application (D) and less activity with
higher synchrony after PDF application (E).
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Results
Fig. 49. PDF induces oscillations. A, B. Extracellular recording from an isolated AME. A. Original recording trace showing
summed action potentials (SAPs) as negative and positive deflections from the baseline. PDF-application (6 pmol via
Picospritzer) induced long-lasting oscillations of the neurons' electrical activity (positive SAPs). Sections with higher SAPamplitudes probably indicate transient synchronization with other assemblies of AME neurons. B. The mean frequency of
the positive SAPs illustrates the oscillations. The data were re-analyzed from (Funk 2005).
3.1.4 8-Br-cAMP but not 8-Br-cGMP mimics all classes of PDF effects
Experiments performed with D. melanogaster demonstrated that the PDFR couples to Gαs and
mediates rises of the second messenger cAMP (Mertens et al. 2005; Shafer et al. 2008; Duvall and
Taghert 2012, 2013). To examine the PDF signal transduction pathway in R. maderae AME neurons it
was tested whether the membrane-permeable and hydrolysis-resistant cAMP analogue 8-Br-cAMP
simulated PDF effects. Additionally, another cyclic nucleotide analogue (8-Br-cGMP) was employed.
8-Br-cAMP was applied at different doses (2 pmol - 1.4 nmol) in 53 recordings, of which 26 were
analyzed. Dissections of the AMAE, used for these experiments, cumulated between ZT 4 and ZT 11
and applications of 8-Br-cAMP between ZT 7 and ZT 12 (Fig. 120). Application of 8-Br-cAMP did not
affect the AME network activity in 66.7 % of the recordings, performed in standard extracellular
saline (n = 16 of 24). In the remaining recordings it caused either an activation (4.2 %, n = 1 of 24,
Fig. 50), inhibitions (16.7 %, n = 4 of 24, Fig. 51, Fig. 55), or an increase in bursting/oscillations
(16.7 %, n = 4 of 24, Fig. 52, Fig. 54, Tab. 7). As shown for PDF, for 8-Br-cAMP multiple simultaneous
effects were observed: In one recording 8-Br-cAMP application caused a transient inhibition and
induced oscillations of the electrical activity. No effects were observed for 8-Br-cAMP applications in
Ca2+-free saline (n = 4 recordings). In the presence of PTX high-amplitude SAPs were elicited in one
recording (n=1 of 2), suggesting the activation or disinhibition of previously silent populations or
transient synchronization of different assemblies (Fig. 123). The delays and durations of the 8-BrcAMP-dependent effects were highly variable: Delays ranged between less than 1 s and more than
26 min and durations ranged between 3 s and more than 2 h. No time-dependency could be
observed (not shown).
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Results
Fig. 50. 8-Br-cAMP activates AME neurons. A, B. Original recording traces of an extracellular recording from an isolated
AME, showing summed action potentials (SAPs) as negative deflections from the baseline. The arrows indicate the
application of 8-Br-cAMP (10 nl, 10 pmol, A) and control solution (10 nl, B) via Picospritzer. While 8-Br-cAMP caused a
transient activation, the control application did not affect the electrical activity of the recorded neurons. The data were reanalyzed from (Funk 2005).
Fig. 51. 8-Br-cAMP inhibits AME neurons. A-C. Extracellular recording from an isolated AME. A. Original recording trace;
the arrow indicates the application of 8-Br-cAMP (100 pmol, via Picospritzer) to the vicinity of the tissue. B, C. Mean
frequency (B) and instantaneous frequency (1/interevent-intervals, C) show, that some of the recorded neurons were
bursting (positive deflections), which was not affected by 8-Br-cAMP. With a delay of 1 s the electrical activity was
inhibited or synchronized to a lower level. The instantaneous frequency band (C) decreased to a lower level. After
approximately 5 min it increased again, and after approximately 21 min it eventually returned to its starting level.
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Results
Fig. 52. 8-Br-cAMP induces oscillations of the AME neurons' electrical activity. A-C. Extracellular recordings from an
isolated AME. A. Original recording trace, showing summed action potentials (SAPs) as positive and negative deflections
from the baseline. The arrow indicates the application of 8-Br-cAMP (200 pmol, via Picospritzer). B. The mean frequency of
the positive SAPs indicates, that 8-Br-cAMP induced long-lasting bursts and oscillations. C. The instantaneous frequency
(1/interevent-interval) plot also illustrates the oscillations. Only in the phases of higher activity a distinct instantaneous
frequency band can be seen, indicating transient synchronization.
The cyclic nucleotide analogue 8-Br-cGMP was applied at different doses (4 - 400 pmol) in
22 recordings, of which 14 were analyzed. The respective dissections cumulated at ZT 4 and between
ZT 7 and ZT 9, and the applications between ZT 6 and ZT 14 (Fig. 120). In contrast to PDF and
8-Br-cAMP, 8-Br-cGMP applications had no effect in the vast majority of recordings in standard
extracellular saline (92.9 %, n = 13 of 14, Fig. 55). In only one recording (7.1 %) the neurons started to
burst for several hours after 8-Br-cGMP application (delay: 9.5 min, ZT 9, Tab. 7, Fig. 53, Fig. 54). In
Ca2+-free saline 8-Br-cGMP did not affect the AME network activity (n = 3 recordings).
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Results
Fig. 53. 8-Br-cGMP induces long-lasting bursts. A-C. Extracellular recording from an isolated AME. A. The grey bar above
the recording trace marks the application of 8-Br-cGMP (a total of 50 pmol, via Picospritzer). B, C. 9 min 30 s after the
application the recorded neurons started bursting with irregular intervals, as can be seen in the mean frequency (B) or
instantaneous frequency (1/interevent-intervals, C) plot. The broad instantaneous frequency band indicates synchronous
firing with similar interevent-intervals (C).
Tab. 7. Effects of PDF, 8-Br-cAMP, and 8-Br-cGMP on the firing rate of neurons of the isolated AME
Activation
Inhibition
Oscillations
No effect
PDF
n=2
n=12
n=7
n=18
5.7 %
34.3 %
20.0 %
51.4 %
8-Br-cAMP
n=1
n=4
n=4
n=16
4.2 %
16.7 %
16.7 %
66.7 %
8-Br-cGMP
n=1
n=13
7.1 %
92.9 %
The sum of recordings, in which each substance was applied, was set 100 %. Multiple effects in some recordings resulted
in a sum of percentages higher than 100 %.
Comparison of the effects of 8-Br-cAMP or 8-Br-cGMP and PDF on the firing rate of AME neurons did
not show a clear simulation of the PDF effects by one of the cyclic nucleotide analogues, although all
classes of PDF effects could be also demonstrated for 8-Br-cAMP (Fig. 54, Fig. 55, Tab. 7). The lack of
effects after 8-Br-cGMP application made cGMP an unlikely second messenger candidate in PDF
signaling, while the effects of 8-Br-cAMP suggested cAMP as second messenger candidate, although
PDF applications resulted in more effects of each category compared to 8-Br-cAMP applications.
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Results
Taking into account only recordings, in which both, PDF and cyclic nucleotide analogues, were
applied, only few recordings (n = 2) were found, in which PDF and 8-Br-cAMP each resulted in an
inhibition (Fig. 55). In the remaining recordings both substances did not affect the neuronal activity
(n = 5), 8-Br-cAMP applications were not analyzable (n = 5), or PDF inhibited while 8-Br-cAMP had no
effect (n = 2). In additional recordings bursting (n = 1) or inhibitions (n = 2) were observed after
application of both PDF and 8-Br-cAMP. Naturally, it was not possible to attribute the effect to one of
the substances. In contrast, there was no recording showing similar effects for PDF and
8-Br-cGMP: Either both substances did not affect the electrical activity (n = 4) or PDF application
resulted in an inhibition while 8-Br-cGMP application had no effect (n = 5).
Fig. 54. Distribution of the effects of PDF, 8-Br-cAMP, and 8-Br-cGMP on the firing rate of AME neurons. The n-numbers
indicate the number of the respective effects. The cyclic nucleotide analogues caused less changes of the firing rate than
PDF. 8-Br-cAMP but not 8-Br-cGMP mimicked all classes of PDF-effects.
Fig. 55. Only 8-Br-cAMP but not 8-Br-cGMP mimics PDF-dependent inhibitions. A-C. Original recording traces of the same
extracellular recording from an isolated AME, showing summed action potentials (SAPs) as negative deflections from the
baseline. The arrows mark the application of 3 pmol PDF (A, re-analyzed from (Funk 2005)), 300 pmol 8-Br-cAMP (B), and
76 pmol 8-Br-cGMP (C) via Picospritzer. While application of PDF and 8-Br-cAMP resulted in a transient inhibition,
8-Br-cGMP application had no effect.
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Results
3.1.5 EPAC might be involved in cAMP-dependent inhibitions of
AME neurons
Since cAMP is a likely candidate for PDF signaling, another cAMP analogue was employed to further
characterize potential targets of cAMP. The cAMP analogue 8-pCPT-2'-O-Me-cAMP (007) was
reported to selectively activate the monomeric G protein EPAC (Enserink et al. 2002). 007 was tested
in ten recordings (at doses of 90 - 310 pmol), of which eight were analyzed. AME-dissections and
experiments were performed at different ZTs (Fig. 120). In 66.6 % of the recordings, performed in
standard extracellular saline (n = 4 of 6) 007 did not affect the network activity, and in 33.3 % of
these recordings (n = 2 of 6, ZT 9 and ZT 21) it inhibited the firing rate (Fig. 56). In recordings
performed in Ca2+-free saline 007 also inhibited the activity (n = 2, ZT 9 and ZT 16). 007-dependent
inhibitions occurred with delays between 4 s and 16.5 min. In no case a regeneration of the electrical
activity was observed. Thus, 007 was more effective than 8-Br-cAMP. In none of the recordings both
007 and PDF were applied.
Fig. 56. EPAC might be involved in cAMP-dependent inhibitions of AME neurons. A, B. Extracellular recording from an
isolated AME. A. Original recording trace, showing summed action potentials (SAPs) as negative deflections from the
baseline. The arrow marks the application of the EPAC-selective cAMP-analogue 8-pCPT-2'-O-Me-cAMP (007, 200 pmol, via
Picospritzer). With a delay of 4 s the electrical activity of the recorded neurons was irreversibly inhibited. B. The inhibition
is also illustrated by the sudden decrease of the mean frequency.
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Results
neurons
While the previous chapter dealt with an examination of the network activity of the central AMEpacemaker of R. maderae, in this chapter the AME neurons were investigated at the level of single
cells. Therefore primary cell cultures of AME neurons (modified from Petri and Stengl 1999) were
prepared and the presumptive pacemaker neurons were used for patch clamp recordings. One
experimental series was performed in ringer solution containing 6 mM CaCl2, which is identical to the
solution used for extracellular recordings of AME explants (3.1, Schneider and Stengl 2005, 2006,
2007), and a second experimental series was performed at a CaCl2 concentration of 1 mM, which was
also used in calcium imaging experiments performed on primary AME cell cultures and shown to
ensure for high spontaneous calcium-activity (Wei 2012; Wei and Stengl 2012; Baz et al. 2013).
3.2.1 Primary AME cell cultures
For the first experimental series 346 cockroaches were sacrificed to prepare primary AME cell
cultures (486 cell cultures from 684 AMAE). The primary cell cultures consisted of neuronal and nonneuronal cells of different size and morphology (Fig. 57). Neurons usually had round somata and
partially extended one or more neurites, which usually did not exceed a length of 50 µm. Nonneuronal cells, often clustered and extended flat, lamellar processes.
Most of the cells died during the first two weeks in vitro, indicating non-optimal culture conditions.
Various strategies to improve the survival rate of the cells were tested: Different cell culture
protocols were used, mainly employing different temperatures and changing the enzymes,
concentrations, and durations for dissociation. Diverse cell culture media were used; amongst others
the medium was supplemented with conditioned medium of a non-neuronal M. sexta cell line (Eide
et al. 1975). Two temperatures were tested for incubation (20°C and 27°C). Cockroaches were
sacrificed at different times after their adult ecdysis. To minimize possible negative influences of
stress hormones, cockroaches were given the possibility to calm down by providing hiding places
after collecting from the breeding colonies. All these attempts failed to improve the survival rate of
the cell cultures. Therefore, morphology and condition of the cell cultures were not further evaluated
and patch clamp experiments generally were performed during the first few days in vitro when the
cells were found to be in their best condition.
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Results
Fig. 57. Primary AME cell cultures contain different cell types. A. Cells of different sizes without arborizations. The arrows
indicate cell debris. B. Neuron sprouted arborizations with varicosities (arrows). C. Neuron with a prominent axon
(arrowhead) and growth cone (arrow). D. Neuron without arborizations (arrowhead) in contact with the patch pipette
(arrow). E. Cell in a bad condition: The bright halo, which characterizes healthy cells, is missing. F. Accumulation of nonneuronal cells (arrow) and a cluster of probably neuronal cells (arrowhead). G, H. Fluorescence images of non-neuronal
cells of different morphology, which were loaded with the calcium sensitive dye fura-2 and excited at 380 nm. Scale bar:
20 µm.
3.2.2 Whole-cell patch clamp recordings of AME neurons reveal
different current components
AME cells with arborizations, identifying them unequivocally as neurons, were chosen preferentially
for patch clamp experiments. The majority of cells died, when an attempt was made to break the
membrane to get into the whole-cell patch clamp mode. The exact proportion of these cells was not
documented. If the whole-cell configuration could be established, the resting membrane potential
(VRMP) of the neurons was measured immediately (Fig. 58). In both experimental series rather high
values were measured (mean ± SD): VRMP = -35.59 mV ± 9.08 mV in 6 mM CaCl2 (n = 34) and
VRMP = -37.75 mV ± 6.97 mV in 1 mM CaCl2 (n = 99). Neither medians (p = 0.3489, Mann Whitney test)
nor means (p = 0.1528, unpaired t-test) significantly differed from each other. Thus, AME neurons in
primary cell culture were depolarized, but the extracellular calcium concentration apparently did not
affect VRMP.
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Results
Fig. 58. The resting membrane potential of AME neurons is not
affected by the extracellular calcium concentration. Box plots with
whiskers (minimum to maximum) showing the distribution of the
resting membrane potential (VRMP), measured immediately after
breaking into the whole-cell configuration. Patch clamp recordings
were performed at an extracellular CaCl2 concentration of 1 mM
(n = 99) or 6 mM (n = 34). Neither medians (p = 0.3489, Mann
Whitney test) nor means (crosses, p = 0.1528, unpaired t-test)
significantly differed from each other (n.s.: not significant).
In the whole-cell mode the AME neurons usually were clamped to -60 mV (holding potential, Vhold)
and stimulated with different voltage-ramp or voltage-step protocols to activate voltage-dependent
currents. Then, current-voltage (I-V) relationships were generated, to analyze the current-responses.
When the neurons were stimulated with voltage-ramp protocols, the corresponding current was
slightly negative at negative voltages (defined as inward current). At its reversal potential (Vrev) the
current turned into a positive current (defined as outward current, Fig. 59). At a CaCl2 concentration
of 1 mM measured values for Vrev ranged from -65 mV to -18 mV and significantly differed,
depending on the respective voltage-ramp protocol (p = 0.0098, one-way ANOVA). At a CaCl2
concentration of 6 mM Vrev ranged from -95 mV to -7 mV, but the values did not significantly differ
depending on the ramp protocols used (p = 0.0572, one-way ANOVA, Tab. 8).
The outward current in response to ramp protocols was multiple times higher than the respective
inward current (defined as outward rectification), indicating that potassium (K+) outward currents (IK)
provide the main component of the whole-cell current (Fig. 59). The outward current continuously
increased with increasing voltage in 61 % of the current traces in response to voltage-ramps (n = 25
of 41) in saline containing 6 mM CaCl2 and in 77 % (n = 37 of 48) in saline containing 1 mM CaCl2
(Fig. 79 I).
Tab. 8. Reversal potential of the whole-cell current
[CaCl2]e
6 mM
6 mM
6 mM
1 mM
1 mM
1 mM
Ramp
+100 mV (100 ms) to -100 mV
+100 mV (40 ms) to -100 mV
-100 mV (-) to +100mV / +140 mV
-100 mV (40 ms) to +100 mV
-110 mV (40 ms) to +90 mV
-140 mV (40 ms) to +80 mV / +100 mV
# of cells
n=24
n=19
n=18
n=10
n=7
n=59
p (normality)
0.9249
0.6930
0.6095
0.3442
n too small
0.4568
Mean
-46.38
-58.05
-48.22
-36.55
-39.71
-44.92
SD
8.546
15.25
23.55
6.551
7.319
8.776
Whole-cell current responses to stimulation with different ramp protocols were analyzed with respect to the reversal
potential Vrev of the current. The duration of a preceding depolarizing or hyperpolarizing voltage step at the starting
potential of the ramp is indicated in brackets. The D'Agostino & Pearson normality test (omnibus K2) was used to confirm
that the data are consistent with a Gaussian distribution. The mean V rev values with standard deviation (SD) were
summarized.
In the remaining recordings (n = 16 of 41, 39 %, at a CaCl2 concentration of 6 mM and n = 11 of 48,
22 %, at a CaCl2 concentration of 1 mM) the outward current only increased to a specific voltage,
then decreased (probably because of an superimposed Ca2+ inward current), and in some cases
eventually increased again, resulting in an "N-shaped" I-V relationship (Fig. 59). However, different
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Results
ramp protocols were used (Tab. 8) and in all 48 cells of the second experimental series voltage ramps
from negative to positive values with preceding hyperpolarization were employed (Fig. 79), which
was not the case for first experimental series.
+
2+
Fig. 59. Interplay between K outward currents and Ca inward currents. A-C. Whole-cell patch clamp recording
performed with an AME neuron in primary cell culture (Ø 15 µm, no arborizations) in saline containing
6 mM CaCl2. The cell was kept at a holding potential of -60 mV and stimulated with a depolarizing voltage ramp from
-100 mV to +140 mV to activate voltage-dependent currents, as shown by the ramp protocol (B). A. The I-V relationship
shows prominent potassium outward currents, which increased until +40 mV, then decreased until +70 mV, and finally
increased again, resulting in a characteristic "N-shape" suggesting the presence of calcium-activated potassium currents
(IK(Ca)). C. The magnification of the detail marked with the dotted line in A shows the reversal potential of the whole-cell
current lying around -38 mV, indicating that small inward currents counteracted the potassium currents at negative
potentials.
When the cells were stimulated with depolarizing voltage-step protocols, more current components
could be distinguished in the responses compared to stimulation with voltage-ramp protocols. Again,
K+ outward currents provided the main component of the whole-cell current in all recordings
(Fig. 60). However, outward currents in response to voltage-steps were always smaller than outward
currents in response to voltage-ramps (Fig. 79). In contrast to the ramp-responses it was possible to
differentiate between transient (fast inactivating) and sustained (non-inactivating) current
components. Therefore, one could differentiate between a transient IA-type-like K+ outward current
(IK,trans), which activated immediately after stimulation and inactivated rapidly, and a sustained
outward current component (IK), which persisted during the complete stimulation (Fig. 60, Tab. 9).
In more than 90 % of the recordings IK did not increase over the complete voltage range of the
respective stimulation protocol (usually -140 mV to +80 mV), as one might expect from solely
voltage-dependent delayed rectifier-type K+ currents (IKdr), but instead reached its peak value at
43.4 mV ± 2.1 mV (n = 67 in 6 mM CaCl2) or 28.7 mV ± 1.8 mV (n = 119 in 1 mM CaCl2), dependent on
the CaCl2 concentration (p < 0.0001, unpaired t-test). At higher voltages IK remained at a plateau,
resulting in a convergence of the corresponding current traces (Fig. 62, Fig. 63, Fig. 124), or
alternatively decreased again, in around 50 % of the recordings even to prominent negative values
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Results
(Fig. 61, Fig. 64), most probably due to a superimposed Ca2+ inward current and/or an involvement of
IK(Ca). Since it could not be differentiated between these possibilities, causing a plateau or a
downward-bend in the I-V relationships of the sustained outward current, this characteristic was
regarded as sustained inward current (Iin,sust), which counteracts IK (see 4.2.3 for a detailed discussion
of the underlying current components). The different manifestations of Iin,sust differed depending on
the CaCl2 concentration (Fig. 61). Additionally, K+ outward currents were present in the so-called tail
currents (Itail), which can be monitored shortly after stimulation and return to Vhold (Fig. 62).
Fig. 60. Sustained potassium outward currents represent the main whole-cell current component of AME neurons. A-D.
Whole-cell patch clamp recording performed with an AME neuron in primary cell culture in saline containing 6 mM CaCl2.
Size and arborizations of the recorded neuron were not documented. A. Voltage step protocol: The cell was kept at a
holding potential of -60 mV and stimulated with depolarizing voltage steps from -140 mV to +80 mV to activate voltagedependent currents. B. A representative current trace shows transient potassium outward current components (IK,trans,
arrow 1), prominent sustained potassium outward current components (I K, arrow 2), a fast, transient sodium inward
current (INa, arrow 3), as well as small sustained inward current components at -140 mV (arrow 4). C. I-V relationships for
the delayed rectifier (IKdr)-like IK (black squares) and the IA-type-like IK,trans (open circles), measured at the positions indicated
by arrows 1 and 2 (in B). D. I-V relationship for the negative peak of the area marked by the bar (arrow 3 in B), showing a
small inward current component at low voltages and a prominent I Na, which activated at -20 mV and peaked at +20 mV. The
reversal potential (Vrev) was more positive than +80 mV.
Tab. 9. Whole-cell current components of AME-neurons
A. 6 mM CaCl2
IK
n=71
100.0 %
B. 1 mM CaCl2
IK
n=124
100.0 %
IK,trans
n=43
60.6 %
INa
n=48
67.6 %
ICa
n=47
66.2 %
Iin,sust
n=67
94.4 %
Itail
n=62
87.3 %
IK,trans
n=78
62.9 %
INa
n=91
73.4 %
ICa
n=81
65.3 %
Iin,sust
n=119
96.0 %
Itail
n=124
100.0 %
~
n=8
11.3 %
~
n=10
8.1 %
Whole-cell patch clamp recordings were performed in saline containing 6 mM CaCl2 (n = 71, A) or 1 mM CaCl2 (n = 124, B).
The neurons were stimulated with voltage step protocols to activate voltage-dependent currents and the current traces
+
were analyzed with respect to the occurring current components. Frequencies of the sustained and the transient K
outward current IK and IK,trans, the transient inward currents INa and ICa, the sustained inward current Iin,sust, and the tail
currents Itail, as well as regularly oscillating outward currents (~) were listed. Percentages relate to the number of cells.
109
Results
2+
Fig. 61. The extracellular Ca -concentration affects the characteristics of Iin,sust. The pie charts illustrate the distribution
of different types of I-V relationships, showing a contribution of the sustained, non-inactivating inward current (Iin,sust),
+
which counteracted voltage-dependent K currents at voltages higher than +20 mV. The respective I-V relationships were
obtained at the beginning of recordings performed in saline containing 6 mM CaCl2 (n = 67) or 1 mM CaCl2 (n = 119).
Strong: The typical downward bend of the I-V curve showed negative values indicating strong, counteracting inward
currents. Moderate: the downward bend of the I-V curve remained positive indicating a moderate counteracting Iin,sust.
Mild: the I-V curve showed only a plateau but not a downward bend, indicating only mild counteracting Iin,sust.
Next to the outward currents and the superimposed Iin,sust prominent transient inward currents could
be identified (Tab. 9). The first transient inward current usually activated faster than all other inward
and outward current components (Fig. 60, Fig. 62). It activated at -37.3 mV ± 8.0 mV (n = 37 in 6 mM
CaCl2, mean ± SD) or -42.3 mV ± 5.6 mV (n = 31 in 1 mM CaCl2) and peaked at +4.3 mV ± 8.5 mV
(6 mM CaCl2) or -7.5 mV ± 8.0 mV (1 mM CaCl2), resembling a sodium (Na+) current (INa). Activation
(p = 0.0052) as well as peak (p < 0.0001, unpaired t-test) were dependent on [CaCl2]e. This current
inactivated in less than 5 ms. A second transient inward current activated and inactivated slightly
slower than INa and superimposed upon IK. It activated at +50.3 mV ± 13.5 mV (n = 38 in 6 mM CaCl2)
or +32.1 mV ± 9.2 mV (n = 28 in 1 mM CaCl2), depending on [CaCl2]e (p < 0.0001, unpaired t-test). It
usually inactivated in less than 10 ms, resembling a transient Ca2+ current (ICa, Fig. 62). ICa increased
with increasing voltage and apparently did not reach its peak within the voltage-range covered by the
stimulation protocols. Additional transient as well as sustained inward current components, most
probably Ca2+ inward currents, were detected in Itail (Fig. 62, Fig. 64). In some recordings (11.3 % in
6 mM CaCl2 and 8.1 % in 1 mM CaCl2) oscillations of the sustained outward currents were detected,
which predominantly occurred at positive potentials (Tab. 9). Only strong, regular oscillations (as
shown in Fig. 72) were counted. The occurrence at positive holding potentials and a NiCl2-dependent
block (Fig. 73) suggested underlying Ca2+ currents.
The composition of the resulting whole-cell current was changed, when the voltage-step protocol
included a preceding hyperpolarizing step (-120 mV or -140 mV). Apparently, the hyperpolarization
always caused the outward current components to increase equally, probably by activation of nonspecific cation currents (Fig. 63, Fig. 64). In no case an exclusive increase of IK,trans was detected, as
expected for an A-type K+ current. Most interestingly, the transient ICa and the non-inactivating Iin,sust
were reduced after hyperpolarizing prepulses, probably due to a superposition of the increased
outward currents. In recordings with a stable ICa in response to step protocols without
hyperpolarization, this current component was completely absent in current traces in response to
step protocols with preceding hyperpolarization (n = 5 in 6 mM CaCl2, n = 30 in 1 mM CaCl2, Fig. 63).
Similarly, the sustained inward current Iin,sust, which counteracted the sustained outward currents,
was reduced or shifted to higher voltages after a preceding hyperpolarization (n = 3 in 6 mM CaCl2,
n = 25 in 1 mM CaCl2, Fig. 64).
110
Results
Fig. 62. AME neurons show two consecutive transient inward current components. A-G. Whole-cell patch clamp recording
from an AME neuron in primary cell culture performed at a CaCl2 concentration of 1 mM. A. Voltage step protocol used to
activate voltage-dependent currents. B. Representative current trace in response to the voltage step protocol.
C. Magnification of the first 10 ms of the current trace, indicated by bar c (in B). Two transient inward current components
(INa and ICa; bar 4 and arrow 5 in C), transient outward currents (IK,trans), sustained outward currents (IK, arrow 1 in B), which
converged at higher voltages, and tail currents (Itail, arrows 2 and 3 in B) can be seen. D. Photograph of the recorded
neuron. Scale bar: 20 µm. E-G. I-V relationships, measured at the positions indicated. E. The transient current (black
squares) consisted of an outward current component activating at -40 mV, which reached its peak at 0 mV (IK,trans), before it
was superimposed by ICa, reaching its highest value at +80 mV. The sustained current (open squares) mainly consisted of a
sustained potassium outward current (IK), which reached a plateau around +60 mV. F. The I-V relationship for the negative
peak of INa (bar 4 in C) shows an activation of the current around -50 mV and a peak at 0 mV. The reversal potential (Vrev) lay
at a voltage more positive than +80 mV, which was not covered by the voltage step protocol. G. The I-V relationship for the
tail currents measured 1 ms (black squares) after return to the holding potential shows a strong outward current
component, which peaked at +30 mV and then declined completely. In contrast, the tail currents measured after 5 ms
(open squares) showed solely a voltage-dependent increase.
111
Results
2+
Fig. 63. Transient Ca current components do not activate after hyperpolarizing pre-pulses. A-H. Whole-cell patch clamp
recording from an AME neuron in primary cell culture performed in saline containing 1 mM CaCl2. A, B. Voltage step
protocols without (A) or with (B) preceding hyperpolarizing pulse (-140 mV). C. Photograph of the recorded neuron (largest
cell). Scale bar: 20 µm. D. Representative current trace in response to the protocol shown in A, showing a prominent,
2+
transient Ca inward current (ICa, bar 4), transient outward currents (IK,trans, arrow 1), sustained outward currents (IK),
which converged at higher voltages (arrow 2), and tail currents (Itail, arrow 3). E. Representative current trace in response
to the protocol shown in B, showing large IK (arrow 5) and Itail (arrow 6), but neither ICa nor prominent IK,trans. Capacitance
artifacts in response to the current changes (arrow 7) indicated non-proper compensation. F-H. I-V relationships measured
at the positions indicated in the legends. F. The sustained outward current elicited without preceding hyperpolarization
reached a plateau at +30 mV. The hyperpolarizing pre-pulse activated additional outward currents causing an increase
over the complete voltage range covered by the protocol. G. I-V relationship for the negative peak of the range indicated
by bar 4 (below ICa in D) showing ICa activating at +20 mV and counteracting IK,trans. H. The I-V relationships for the tail
currents, measured without preceding hyperpolarization (black squares), show a downward bend of the outward current,
while the outward tail currents measured with preceding hyperpolarization (open circles) apparently reached a plateau at
+80 mV without showing a decrease at higher voltages.
112
Results
Fig. 64. Hyperpolarizing pre-pulses activate additional sustained current components. A-F. Whole-cell patch clamp
recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A, B. Voltage step
protocols without (A) or with (B) preceding hyperpolarizing pulse (-140 mV). C. Photograph of the recorded neuron. Scale
bar: 20 µm. D. Representative current trace in response to the protocol shown in A, showing large, sustained potassium
2+
outward currents (IK, arrow 1), large transient Ca inward currents (ICa, arrow 3), strong sustained inward currents (Iin,sust,
arrow 4), as well as prominent tail currents (Itail, arrow 2). E. Representative current trace in response to the protocol shown
in D, showing large outward current (arrow 5) and Itail (arrow 6) components. F, G. Representative I-V relationships
measured at the positions indicated. F. Without preceding hyperpolarization (black squares) IK activated at -40 mV and
increased to its peak value at +20 mV, before Iin,sust counteracted, resulting in a decrease of the whole-cell current to
negative values. With hyperpolarizing pre-pulse (open circles) an inward current component could be detected at
potentials more negative than -40 mV. The sustained outward current was increased, and the counteracting Iin,sust activated
at a more positive voltage (+80 mV), suggesting the activation of superposing outward currents. G. The same applies for the
I-V relationships for the tail currents, measured 1 ms after return to the resting potential.
3.2.3 Development of the whole-cell current components during a
recording
Most whole-cell recordings lasted only a few minutes, before the cells were lost. This process was
accompanied by a darkening of the usually bright cell (Fig. 57 E) or shrinkage of the cell, which could
not be prevented by an adjustment of the recording solutions' osmolarity. Different osmolarities of
113
Results
the pipette solution were tested but did not solve the problem (not shown). Exact durations of
recordings and proportions of cells were not analyzed. To evaluate the stability of the current
components during a recording, recordings that lasted longer than 4 min and did not involve
pharmacological treatment were analyzed (n = 15 cells in 6 mM CaCl2; n = 14 cells in 1 mM CaCl2,
Tab. 10). The current traces in response to voltage-step protocols, the corresponding I-V
relationships, and particularly the time courses of the area under the I-V curves or the time courses
of the peak currents clearly revealed that the analyzed current components usually did not remain
stable during the recording. The fast transient inward current INa was the only component that
showed no change in a few cases. In the vast majority of recordings IK as well as the different inward
current components spontaneously decreased in an almost linear or exponential manner (Fig. 65).
Only in rare cases current components increased or showed phases of decrease as well as phases of
increase during one recording. Interestingly, the sustained inward current that counteracted IK (Iin,sust)
often passed through all stages of its expression while decreasing, starting with prominent negative
values at higher voltages in the beginning of the recording and ending with a very low expression,
which caused IK to reach a plateau or to show a pure voltage-dependent increase (Fig. 66).
Tab. 10. Spontaneous changes of whole-cell current components in control recordings
A. 6 mM CaCl2
Decrease
Increase
Increase + decrease
No change
# cells
IK
n=12
80.0 %
n=3
20.0 %
INa
n=12
n=1
n=13
n=15
92.3 %
ICa
n=8
100.0 %
7.7 %
n=8
Iin,sust
n=12
n=1
n=1
n=1
n=15
80.0 %
6.7 %
6.7 %
6.7 %
B. 1 mM CaCl2
Decrease
Increase
Increase + decrease
No change
Not analyzable
# cells
IK
n=12
85.7 %
INa
n=7
63.6 %
n=1
n=1
7.1 %
7.1 %
n=4
36.4 %
n=14
n=11
ICa
n=7
77.8 %
n=1
11.1 %
n=1
n=9
11.1 %
Iin,sust
n=7
n=2
n=2
n=1
58.3 %
16.7 %
16.7 %
8.3 %
n=12
Whole-cell patch clamp recordings with durations longer than 4 min (n = 15, A and n = 14, B) were analyzed with respect
to spontaneous changes without preceding pharmacological treatment. Changes in different whole-cell current
components, such as the sustained potassium outward current I K, the transient inward currents INa and ICa, and the
sustained inward current Iin,sust were summarized. Percentages relate to the number of cells (# cells) expressing the
respective current component.
114
Results
Fig. 65. Several current components spontaneously decreased in the course of the recordings. A-I. Whole-cell patch clamp
recording from an AME neuron in primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step protocol
used to activate voltage-dependent currents. B. Current traces, recorded in the beginning of the recording (top) and after
400 s (bottom). A decrease of several current components, such as the transient outward current (IK,trans, arrow 1), the
sustained outward current (IK, arrow 2), the tail currents (Itail, arrow 3), as well as the inward currents (INa and ICa, arrows 4
and 5) can be seen. C. Photograph of the recorded neuron. Scale bar: 20 µm. D-F. I-V relationships for all protocol runs of
the recording for IK (D, measured at the position indicated by arrow 2 in B), for the negative peak of INa alone (E, measured
over the range indicated by bar e in B), and for the negative peak of both transient inward currents (F, measured over the
range indicated by bar f in B). All current components decreased during the recording. While the first I-V curves for IK (D)
show a downward bend at +80 mV, this is not the case for all subsequent curves, which all reach a plateau at higher
voltages but do not decrease. G-I. The time courses for the areas under the respective I-V curves (AUC) for IK (G), INa alone
(H), and both transient inward currents (I) also illustrate the spontaneous decrease of these current components.
115
Results
2+
+
Fig. 66. Rundown of Ca inward currents and interaction with K outward currents. A-E. Whole-cell patch clamp
recording from an AME neuron (Ø 22 µm, no arborizations) performed in saline containing 1 mM CaCl2. A-C. Current traces
in response to the voltage step protocol (shown in D) at different times of the recording. The arrow indicates the position,
at which I-V relationships (E) were generated. The symbols next to the current traces indicate the respective I-V curves (E).
E. I-V relationships obtained after 4 s (black squares), 64 s (black circles), 205 s (black triangles), 437 s (open, upside-down
2+
triangles), and 497 s (open circles). At the beginning of the recording presumptive non-inactivating Ca inward currents
2+
+
+
(Iin,sust) were present, which activated Ca -dependent K currents and counteracted the voltage-dependent K outward
+
currents at voltages higher than +20 mV. At the end of the recording voltage-dependent K outward currents constituted
2+
2+
+
the main part of the whole-cell current, probably due to a rundown of Ca currents and Ca -dependent K currents.
3.2.4 Pharmacological characterization of the whole-cell currents
As shown before, all current components in control recordings without pharmacological treatment
spontaneously decreased in most cases. Additional control recordings were performed, in which
control solution (= bath solution without any pharmacological supplements) was applied (n = 4 in
1 mM CaCl2). In most cases application of control solution did not affect the cell (Tab. 11, Fig. 67, Fig.
125), but in one recording IK, INa, and ICa decreased apparently application-dependently (not shown).
Next, different ion channel blockers were employed to further characterize the different current
components. To evaluate possible effects of the drugs, the time courses of the areas under the
respective I-V curves or the time courses of respective peak currents were analyzed. Only if changes
appeared to correlate with the application they were accounted as effects. All drugs were used in
concentrations, which previously were shown to be effective in calcium imaging experiments on AME
neurons (Wei and Stengl 2012; Wei et al. 2014).
Different K+ channel blockers were employed. When tetraethylammonium (TEA), a rather unspecific
K+ channel blocker, was applied to the extracellular solution (20 mM, n = 15), transient and sustained
K+ currents as well as the counteracting Iin,sust and the transient ICa were reduced. In contrast, the fast
116
Results
INa was increased or unaffected (Tab. 12, Fig. 68, for a separation of the effects by the CaCl2
concentration see Tab. 25).
Tab. 11. Effects of control applications
IK
n=1
n=1
n=2
Decrease
No effect
Uncorrelated decrease
Not analyzable
# cells
25.0 %
25.0 %
50.0 %
n=4
INa
n=1
n=3
25.0 %
75.0 %
n=4
ICa
n=1
n=1
n=1
Iin,sust
33.3 %
33.3 %
33.3 %
n=3
n=1
50.0 %
n=1
n=2
50.0 %
In four whole-cell patch clamp recordings ([CaCl2]e = 1 mM) control applications (saline) were performed. Effects on
different whole-cell current components, such as the sustained potassium outward current I K, the transient inward
currents INa and ICa, and the sustained inward current Iin,sust were summarized. Percentages relate to the number of cells (#
cells) expressing the respective current component. Uncorrelated decrease: decrease, which started before application
and was not affected by it.
Tab. 12. Effects of TEA application
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=13
86.7 %
IK,trans
n=12
85.7 %
n=2
13.3 %
n=1
n=1
7.7 %
7.7 %
n=15
n=14
INa
n=1
n=5
n=4
n=10
10.0 %
50.0 %
40.0 %
ICa
n=6
n=2
n=8
75.0 %
Iin,sust
n=6
n=3
25.0 %
n=1
n=5
n=15
40.0 %
20.0 %
6.7 %
33.3 %
In 15 whole-cell patch clamp recordings tetraethylammonium (TEA) was bath-applied. Effects on different whole-cell
current components, such as the sustained potassium outward current IK, the transient outward current IK,trans, the
transient inward currents INa and ICa, and the sustained inward current Iin,sust were summarized. Percentages relate to the
number of cells (# cells) expressing the respective current component. Uncorrelated decrease: decrease, which started
before application and was not affected by it.
117
Results
Fig. 67. Application of control solution did not affect the whole-cell current components. A-G. Whole-cell patch clamp
recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step
protocol used to activate voltage-dependent currents. B, C. Representative current traces before (B) and after (C)
perfusion with bath solution without any additional ingredients (control solution) showing transient outward currents
(IK,trans, arrow1), sustained outward currents (IK, arrow 2), tail currents (Itail, arrow 3), the first transient inward current (INa,
arrow 4), and the second transient inward current (ICa, arrow 5). D. Photograph of the recorded neuron. Scale bar: 20 µm.
E-G. I-V relationships for the sustained outward current (E, measured at the position indicated by arrow 2 in B), for the
transient currents (F, arrow 1), for the mean Itail (G, measured over the range indicated with bar g), and for the negative
peak of INa (H, measured over the small range indicated by bar h), measured before (black squares) or after perfusion with
control solution (open squares). The sustained IK (E), the transient IK,trans (F), as well as the transient ICa (F) showed a small,
application-independent reduction (see Fig. 125 for more information), while Itail (G) and INa (H) remained stable. Data for
the I-V relationships are given as means ± SEM (n = 3 protocol runs).
118
Results
Fig. 68. TEA blocks sustained potassium outward currents. A-G. Whole-cell patch clamp recording from an AME neuron in
a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step protocol used to activate voltagedependent currents. B, C. Representative current traces before (B) and after (C) perfusion (for 2 min) with the potassium
channel blocker tetraethylammonium (TEA, 20 mM) showing a reduction of the sustained outward current (I K, arrow 1) and
+
the tail current (Itail, bar g). In contrast, the transient outward current (IK,trans, arrow 4) and the fast transient Na current INa
(bar h) were increased. D. Photograph of the recorded neuron. Scale bar: 20 µm. E-H. I-V relationships for IK (E, measured at
the position indicated by arrow 1 in B), for IK,trans (F, arrow 4 in C), for the mean Itail (G, measured over the range indicated by
bar g in B), and for the negative peak of INa (H, bar h in B) before (black squares) and after (open squares) TEA application.
While TEA blocked IK and the downward bend at voltages higher than +20 mV (E) as well a Itail (G), it intensified and
accelerated IK,trans (F) and increased INa (H). Data for the I-V relationships before TEA application are given as means ± SEM
(n = 3 protocol runs). The data were re-analyzed from (Yasar 2013).
119
Results
The K+ channel blocker CsCl was applied via the pipette solution, where it substituted for KCl
(160 mM, n = 6 recordings). In these recordings, the CsCl-dependent block of K+ currents revealed
new inward current components, which have never been observed with standard pipette solution.
Instead of the fast INa and the somewhat slower ICa (Fig. 62) another Ca2+ inward current with a fast,
inactivating component (ICa,trans(Cs)) and a sustained, non-inactivating component (ICa,sust(Cs)) were
detected (Fig. 69, Fig. 74). Both components activated around -50 mV, peaked around -10 mV, and
were NiCl2-sensitive. Whether CsCl was applied in combination with TEA (n = 1) or alone (n = 5), in all
recordings residual outward currents (Iout,res) were detected, indicating an incomplete block of K+
currents or the presence of other outward currents, probably non-specific cation currents or chloride
currents (ICl, caused by influx of Cl- ions).
Fig. 69. Block of outward potassium currents reveals strong inward current components. A-E. Whole-cell patch clamp
recording from an AME neuron in primary cell culture performed in saline containing 1 mM CaCl2. Potassium currents were
blocked by substitution of KCl with CsCl (160 mM) in the pipette solution and addition of TEA (20 mM) to the extracellular
solution. A. Photograph of the recorded neuron. Scale bar: 20 µm. B. Voltage step protocol used to activate voltagedependent currents. C. Representative current trace in response to the voltage step protocol shown in B. Transient (arrow
1) and sustained outward current components (arrow 2), as well as transient (bar e) and sustained inward current
components can be seen. D. The I-V relationships for the transient currents (open circles), measured at the position
indicated by arrow 1 in C, and the sustained current (black squares, arrow 2 in C) both show an inward current component
peaking at -20 mV and an outward current component, which increased until +30 mV and then decreased again. E. The I-V
relationship for the negative peak of the transient inward current (measured over the range indicated by bar e in C) shows
an activation around -60 mV and a peak at -20 mV. Additionally, another small inward current component at lower
voltages becomes apparent. The data were re-analyzed from (Yasar 2013).
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Results
Next, the membrane-permeable K+ channel blocker 4-aminopyridine (4-AP) was used. It was added in
a concentration of 1 mM or 4 mM to the extracellular solution (n = 13 recordings). 4-AP was not as
effective as TEA, but in approximately one third of the recordings all current components
investigated (IK, IK,trans, INa, ICa, and Iin,sust) were reduced 4-AP-dependently (Tab. 13, Fig. 70, for a
separation of the effects by the CaCl2 concentration see Tab. 26).
To analyze whether IK(Ca) contributed to the whole-cell outward currents, apamin was employed,
which blocks small conductance Ca2+-activated K+ channels (SK). Apamin was applied in recordings
performed in 1 mM CaCl2 in concentrations of 1 mM or 1 µM (n = 5 recordings). An applicationdependent decrease of IK was detected only in the single recording, in which the higher
concentration was employed. ICa and Iin,sust decreased in two recordings, Itail in three recordings. INa
was not affected by apamin (Tab. 14, Fig. 71).
Tab. 13. Effects of 4-AP application
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=4
n=2
n=2
n=5
30.8 %
15.4 %
15.4 %
38.5 %
n=13
IK,trans
n=5
n=2
n=1
n=4
n=1
n=13
38.5 %
15.4 %
7.7 %
30.8 %
7.7 %
INa
n=3
30.0 %
n=4
n=2
n=1
n=10
40.0 %
20.0 %
10.0 %
ICa
n=4
n=2
n=2
n=4
33.3 %
16.7 %
16.7 %
33.3 %
n=12
Iin,sust
n=3
n=1
n=5
n=1
n=10
30.0 %
10.0 %
50.0 %
10.0 %
In 13 whole-cell patch clamp recordings 4-aminopyridine (4-AP, 1 mM or 4 mM) was bath-applied. Effects on different
whole-cell current components, such as the sustained potassium outward current I K, the transient outward current IK,trans,
the transient inward currents INa and ICa, and the sustained inward current Iin,sust were summarized. Percentages relate to
the number of cells (# cells) expressing the respective current component. Uncorrelated decrease: decrease, which started
before application and was not affected by it.
Tab. 14. Effects of apamin application
Decrease
No effect
Uncorrelated
decrease
Not analyzable
# cells
IK
n=1
n=1
n=3
n=5
INa
20.0 %
20.0 %
60.0 %
n=2
n=1
n=3
66.7 %
ICa
n=2
n=1
n=2
Iin,sust
n=2 40.0 %
40.0 %
20.0 %
40.0 %
n=3
33.3 %
n=5
n=5
2+
Iout,tail
n=3 60.0 %
n=1 20.0 %
60.0 %
n=1
n=5
20.0 %
Iin,tail
n=1
25.0 %
n=2
50.0 %
n=1
n=4
25.0 %
In five whole-cell patch clamp recordings the small conductance, Ca -dependent potassium channel (SK) blocker apamin
(1 µM or 1 mM) was bath-applied ([CaCl2]e = 1 mM). Effects on different whole-cell current components, such as the
sustained potassium outward current IK, the transient inward currents INa and ICa, the sustained inward current Iin,sust, and
the tail currents Iout,tail and Iin,tail were summarized. Percentages relate to the number of cells (# cells) expressing the
respective current component. Uncorrelated decrease: decrease, which started before application and was not affected by
it.
121
Results
Fig. 70. 4-AP blocks inward and outward current components. A-G. Whole-cell patch clamp recording from an AME neuron
in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step protocol used to activate voltagedependent currents. B, C. Representative current traces before (B) and after (C) perfusion (2 min) with the potassium
channel blocker 4-aminopyridine (4-AP, 4 mM) showing a reduction of the transient outward current (IK,trans, arrow 1), the
sustained outward current (IK, arrow 2), the tail currents (Itail, bar h), INa (arrow 3), and ICa (arrow 4). D. Photograph of the
recorded neuron. Scale bar: 20 µm. E-H. I-V relationships for IK (E, measured at the position indicated by arrow 2 in B), for
the transient current (F, arrow 1 in B), for the mean Itail (G, measured over the range indicated by bar g in B), and for the
negative peak of INa (H, bar h in B), measured before (black squares) or after application of 4-AP (open squares). 4-AP
reduced the outward current components IK (E) and IK,trans (F) as well as the fast INa (h) and the slower ICa (F). The reduction
of the outward current can also be seen in the tail currents (G). Furthermore, 4-AP blocked the downward bend of the I-V
curves at voltages higher than +30 mV or +40 mV, which becomes apparent in IK (E) and Itail (G). Data for the I-V
relationships are given as means ± SEM (n = 3 protocol runs).
122
Results
Fig. 71. Apamin blocks inward and outward current components. A-G. Whole-cell patch clamp recording from an AME
neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step protocol used to activate
voltage-dependent currents. B, C. Representative current traces before (B) and after (C) application of the potassium
channel (SK) blocker apamin (300 µl, 1 mM) show a reduction of the sustained outward current (IK, arrow 1), the tail current
(Itail, bar f), the second transient inward current ICa (arrow 3), and the sustained inward current (Iin,sust, arrow 4), but not INa
(arrow 2). D. Photograph of the recorded neuron. Scale bar: 20 µm. E-G. I-V relationships for IK (E, measured at the position
indicated by arrow 1 in B), for the mean Itail (F, measured over the range indicated by bar f in B), and for the negative peak
of INa and ICa (H, bar g in B), measured before (black squares) and after application of apamin (open squares). Apamin
application reduced IK (E), Itail (F), as well as ICa (G), while the fast INa peaking at -20 mV (G) was not affected. Additionally,
apamin blocked Iin,sust, responsible for the downward bend of the I-V curves at +80 mV, which becomes apparent in the
sustained current (E) and Itail (F). Data for the I-V relationships are given as means ± SEM (n = 3 protocol runs).
For a pharmacological characterization of the inward current components the Na+ channel blocker
TTX and the Ca2+ channel blockers NiCl2 and mibefradil were employed. The fast transient inward
current (INa) was blocked by TTX (10 nM) in all recordings (n = 15 in 1 mM CaCl2) confirming the
identity of this current. In addition TTX also blocked ICa and increased Iin,sust in about one third of the
recordings showing these current components (Tab. 15, Fig. 72, Fig. 126).
NiCl2, a rather unspecific blocker of Ca2+ channels, was applied to the bath solution, where it
completely or incompletely substituted for CaCl2. In recordings performed in 1 mM CaCl2, NiCl2
completely substituted for CaCl2 (1 mM). In recordings performed at a CaCl2 concentration of 6 mM,
CaCl2 was partially (2 mM CaCl2, 4 mM NiCl2) or fully (6 mM) replaced by NiCl2. All concentrations
were equally effective and had multiple effects on the analyzed current components. When NiCl2 was
applied in the absence of other blockers (n = 12 in 1 mM CaCl2), ICa was blocked in 80 % of the cells
that expressed this component (Tab. 16). Iin,sust was also affected in 80 % of the cells, splitting in 30 %
showing a decrease and 50 % showing an increase, indicating a direct or indirect Ca2+-dependence of
this current. IK was reduced in more than half of the cells and INa in almost one third of the cells
(Tab. 16, Fig. 73).
123
Results
Tab. 15. Effects of TTX application
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=2
n=1
n=4
n=3
n=3
n=13
15.4 %
7.7 %
INa
n=13
100.0 %
23.1 %
23.1 %
ICa
n=3
n=1
n=3
n=3
n=10
n=13
30.0 %
10.0 %
30.0 %
30.0 %
Iin,sust
n=1
n=3
n=1
n=4
n=1
n=10
10.0 %
30.0 %
10.0 %
40.0 %
10.0 %
In 13 whole-cell patch clamp recordings tetrodotoxin (TTX, 10 nM) was applied ([CaCl 2]e = 1 mM). Effects on different
whole-cell current components, such as the sustained potassium outward current IK, the transient inward currents INa and
ICa, and the sustained inward current Iin,sust were summarized. Percentages relate to the number of cells (# cells) expressing
the respective current component. Uncorrelated decrease: decrease, which started before application and was not
affected by it.
When NiCl2 was applied while K+ channels were blocked with CsCl in the pipette solution (n = 5 in
6 mM CaCl2), all current components observed in the presence of CsCl were reduced (Tab. 17,
Fig. 74). The residual outward current (Iout,res) and Iin,sust decreased in 60 % of the recordings and the
transient (ICa,trans(Cs)) as well as the sustained Ca2+ current component (ICa,sust(Cs)) in all recordings.
Interestingly, the NiCl2-dependent block of these Ca2+ current components revealed another small
inward current component, which was masked by ICa,trans(Cs) before (n = 1). This current was shown to
be TTX-sensitive, indicating a Na+ current (Fig. 74).
The last Ca2+ channel blocker that was tested was mibefradil (n = 7 in 1 mM CaCl2). It was shown to
be less effective than NiCl2. Mibefradil reduced IK and ICa in around one third of the cells and
increased Iin,sust in 42.9 % of the cells (n = 3 of 7 cells, Tab. 18, Fig. 75).
Tab. 16. Effects of NiCl2 application
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=7
58.3 %
n=5
41.7 %
n=12
INa
n=2
n=1
n=4
n=7
28.6 %
14.3 %
57.1 %
ICa
n=8
n=1
n=1
n=10
80.0 %
10.0 %
10.0 %
Iin,sust
n=3
n=5
30.0 %
50.0 %
n=2
20.0 %
n=10
In twelve whole-cell patch clamp recordings the extracellular solution containing 1 mM CaCl2 was exchanged with
extracellular solution containing 1 mM NiCl2. Effects on different whole-cell current components, such as the sustained
potassium outward current IK, the transient inward currents INa and ICa, and the sustained inward current Iin,sust were
summarized. Percentages relate to the number of cells (# cells) expressing the respective current component.
Uncorrelated decrease: decrease, which started before application and was not affected by it.
124
Results
+
Tab. 17. Effects of NiCl2 application during CsCl-dependent block of K outward currents
Decrease
Not analyzable
# cells
Iout,res
n=3
n=1
n=1
n=5
60.0 %
20.0 %
20.0%
ICa,trans(Cs)
n=5
100.0 %
ICa,sust(Cs)
n=4
100.0 %
Iin,sust
n=3
n=2
n=5
n=4
n=5
60.0 %
40.0 %
In five whole-cell patch clamp recordings the extracellular solution containing 6 mM CaCl 2 was exchanged with
extracellular solution containing 4 mM NiCl2 and 2 mM CaCl2 or solely 6 mM NiCl2. The intracellular solution contained
160 mM CsCl instead of KCl. Effects on different whole-cell current components, such as the residual, sustained potassium
outward current Iout,res, the transient inward current ICa,trans, and the sustained inward current ICa,sust were summarized.
Percentages relate to the number of cells (# cells) expressing the respective current component. Uncorrelated decrease:
decrease, which started before application and was not affected by it.
Tab. 18. Effects of mibefradil application
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=2
28.6 %
n=1
n=4
14.3 %
57.1 %
n=7
INa
n=1
16.7 %
n=3
50.0 %
n=2
n=6
33.3 %
ICa
n=2
n=1
n=2
n=1
n=6
33.3 %
16.7 %
33.3 %
16.7 %
Iin,sust
n=1
n=3
n=2
n=1
n=7
14.3 %
42.9 %
28.6 %
14.3 %
In seven whole-cell patch clamp recordings mibefradil (10 µM) was applied (1 mM CaCl2). Effects on different whole-cell
current components, such as the sustained potassium outward current IK, the transient inward currents INa and ICa, and the
it.
Finally, the effects of GABA on the whole-cell current components of AME cells were examined (n = 2
recordings in 6 mM CaCl2). GABA was applied via pipette (1 nmol) and increased a sustained inward
current component at voltages lower than -100 mV (n = 1) and the sustained outward current (n = 2)
in current responses to voltage-step protocols (not shown). The increase of the inward current and
the outward current could be better observed, when the cell was stimulated with ramp protocols
(n = 1). In this case, subtraction of the current responses revealed the GABA-dependent current
(Fig. 76), which was almost linear and outward rectifying with a reversal potential of -50 mV. The
reversal potential was not changed by GABA application. Most probably this current was a Cl- current
(ICl), suggesting that ICl also contributed to the observed outward currents in other recordings.
125
Results
Fig. 72. TTX blocks the fast transient inward current component. A-G. Whole-cell patch clamp recording from an AME
neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step protocol: The cell was kept at a
holding potential of -60 mV and stimulated with depolarizing voltage steps from -140 mV to +80 mV to activate voltagedependent currents. B, C. Representative current traces before (B) and after (C) perfusion (for 2 min) with the sodium
+
channel blocker tetrodotoxin (TTX, 10 nM) showing a reduction of the transient outward current (IK,trans, bar f) and the Na
inward current INa (bar h). Oscillations of the currents as well as the sustained outward current (I K, arrow) and the tail
current (Itail, bar g) apparently were not affected. D. Photograph of the recorded neuron. Scale bar: 20 µm. E-H. I-V
relationships for IK (E, measured at the position indicated by the arrow in B), for IK,trans (F, positive peak of the range
indicated by bar f), for the mean Itail (G, range indicated by bar g), and for INa (H, negative peak of the range indicated with
bar h) before (black squares) and after TTX application (open squares). While IK,sust (E) and Itail (G) were not affected, INa was
clearly blocked by TTX (H). IK,trans decreased spontaneously and independently of TTX application (F), confirmed by
comparison with the time course of the peak current (not shown). Data for the I-V relationships are given as means ± SEM
(n = 3 protocol runs).
126
Results
Fig. 73. NiCl2 blocks inward and outward current components as well as oscillations. A-G. Whole-cell patch clamp
recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step
protocol: The cell was kept at a holding potential of -60 mV and stimulated with depolarizing voltage steps from -140 mV to
+80 mV to activate voltage-dependent currents. B, C. Representative current traces before (B) and after (C) perfusion
(2 min) with extracellular solution containing NiCl2 instead of CaCl2 (1 mM) showing a reduction of the transient outward
current (IK,trans, arrow 1), the sustained outward current (IK, arrow 2), the tail current (Itail, bar g), the first transient inward
current (INa, arrow 3) and the second transient inward current ICa (arrow 4). Next to these current components small
oscillations in the sustained outward currents at positive potentials were blocked. D. Photograph of the recorded neuron
(arrow). Scale bar: 20 µm. E-H. I-V relationships for IK (E, measured at the position indicated by arrow 2 in B), for the
transient currents (F, arrow 1), for the mean Itail (F, measured over the range indicated by bar g), and for the negative peak
of INa (H, measured over the range indicated by bar h), measured before (black squares) and after perfusion with NiCl2
(open squares). NiCl2 reduced the outward current components IK (E) and IK,trans (F) as well as the fast INa (H) and the slower
ICa (inward current component in F). The reduction of the outward current can also be seen in I tail (G). Next to these current
reductions, NiCl2 apparently caused IK to decrease after reaching its peak value at +20 mV (E). The resulting downward bend
of the I-V curve after NiCl2 application becomes also apparent in the tail current at voltages higher than 0 mV (G). Data for
the I-V relationships are given as means ± SEM (n = 3 protocol runs).
127
Results
+
Fig. 74. The transient inward current in the presence of Cs consists of ICa and INa components. A-J. Whole-cell patch
+
clamp recording from an AME neuron in a primary cell culture performed in saline containing 6 mM CaCl2. K channels
were particularly blocked by CsCl (160 mM) in the pipette solution. A. Voltage step protocol used to activate voltagedependent currents. D. Photograph of the recorded neuron (arrow). Scale bar: 20 µm. B, C, E, F. Representative current
traces under control conditions (B), in the presence of 6 mM NiCl2 (C), NiCl2 and tetrodotoxin (TTX, 10 nM, E), and after
washout (F). NiCl2 reduced the transient (IK,trans, arrow 1 in B) and the sustained outward current (IK, arrow 2), the tail
2+
currents (Itail, arrow 3), and the transient and sustained Ca inward current components (ICa, arrow 4). The NiCl2dependent reduction of ICa revealed another small inward current component (INa, arrow 5 in C), which was TTX-sensitive
(E). After washout of NiCl2 and TTX, all current components slightly recovered (F). G-J. The I-V relationships for IK (G,
measured at the position indicated by arrow 2 in B), for the transient currents (H, arrow 1), for the mean Itail (I, measured
over the range indicated by bar i), and for the negative peak of the transient inward currents (J, bar j) illustrate the NiCl2and TTX-dependent reduction and the partial recovery after washout (G-J). Data for the I-V relationships are given as
means ± SEM (n = 5 protocol runs before treatment, n = 7 in the presence of NiCl2, n = 8 in the presence of NiCl2 and TTX,
n = 3 after washout).
128
Results
2+
Fig. 75. Mibefradil blocks the transient Ca inward current and increases a sustained inward current component. A-G.
Whole-cell patch clamp recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2.
A. Voltage step protocol: The cell was kept at a holding potential of -60 mV and stimulated with depolarizing voltage steps
from -140 mV to +80 mV to activate voltage-dependent currents. B, C. Representative current traces before (B) and after
(C) perfusion (for 3 min) with the calcium channel blocker mibefradil (10 µM) showing a block of the second transient
inward current (ICa, arrow 4 in B) as well as an activation of a new sustained inward current (Iin,sust, arrow 5 in C)
counteracting the sustained outward currents (IK, arrow 2 in B). D. Photograph of the recorded neuron. Scale bar: 20 µm.
E-G. I-V relationships for IK (E, measured at the position indicated by arrow 2 in B), for the transient currents (F, arrow 1),
and for the mean Itail (G, measured over the range indicated by bar g), before (black squares) and after mibefradil
application (open circles). The mibefradil-dependent downward bend of the I-V curves for IK (E) and Itail (G) at voltages
higher than +20 mV illustrates the activation of Iin,sust. The reduction of the inward current component in F illustrates the
block of the transient ICa. Data for the I-V relationships are given as means ± SEM (n = 3 protocol runs).
129
Results
-
Fig. 76. GABA elicits a presumptive Cl current. A-D. Whole-cell patch clamp recording from an AME neuron (Ø 15 µm, no
arborizations) in a primary cell culture performed in saline containing 6 mM CaCl2. A. Voltage-ramp protocol: The cell was
kept at a holding potential of -60 mV, depolarized to +100 mV for 100 ms and then hyperpolarized to -100 mV, before
turning back to -60 mV. B, C. Current traces in response to the ramp protocol before (B) and after (C) pipette application of
1 nmol γ-aminobutyric acid (GABA). D. I-V relationships for the current traces before (light grey) and after GABA
application (dark grey), and for the GABA-dependent current (black), obtained by subtracting the current before GABA
from the current in the presence of GABA. GABA application resulted in higher inward current and higher, more linear
outward current, possibly via GABAA-receptor mediated Cl -influx. The reversal potential (-50 mV) was not changed.
3.2.5 Effects of PDF on different current components of AME
neurons
The next experiments aimed at the analysis of the effects of the neuropeptide pigment-dispersing
factor (PDF), which was previously shown to synchronize different assemblies of AME neurons via
inhibition and activation/disinhibition of their electrical activity (Schneider and Stengl 2005) and to
induce oscillations of the electrical activity, probably via cAMP rises (see 3.1.3 and 3.1.4). PDF was
applied via pipette (1 pmol - 0.25 nmol), pressure ejection (Picospritzer, 1 fmol - 1 pmol) or the
perfusion system (500 nM) in recordings performed in saline containing 6 mM CaCl2 (n = 9) or
1 mM CaCl2 (n = 37). All concentrations used were previously shown to be effective in extracellular
recordings (Schneider and Stengl 2005) or calcium imaging experiments performed with AME
neurons (Wei et al. 2014; Baz 2015).
PDF affected all current components that were analyzed, whereas current reductions were the
predominant effects (Tab. 19, Fig. 77, Fig. 78, Fig. 79) and current increases only rarely were
detected (Tab. 19, Fig. 79, Fig. 80, for a separation of the effects by [CaCl2]e see Tab. 27). PDF
decreased IK in 21.7 % of the cells (Fig. 77, Fig. 79, Fig. 126), INa in 14.3 % of the cells (Fig. 77, Fig.
126), ICa in 30 % of the cells (Fig. 77, Fig. 126), and Iin,sust in 18.4 % of the cells (Fig. 78). In one
recording, where PDF decreased the fast transient INa, washing out PDF with saline containing TTX
(10 nM) almost completely decreased the residual fast transient inward current, indicating that this
current component indeed was INa (Fig. 126). In contrast PDF increased IK in only 4.3 %, INa in 5.7 %, ICa
in 13.3 % and Iin,sust in 7.9 % of the recordings showing the respective current components (not
130
Results
shown). In 32.6 % of the recordings (n = 14 of 43) PDF activated an inward current at negative
potentials, probably a non-specific cation current such as Ih (Fig. 79). This current could be observed
in current traces in response to stimulation with ramp protocols at voltages lower than Vrev. It caused
Vrev to shift to more positive values in 50 % (n = 7 of 14 recordings), or did not affect Vrev in 42.9 %
(n = 6 of 14 recordings), while a shift to more negative values was observed only in one recording.
All these effects of PDF were observed within several minutes after PDF application. Usually PDF
effects were long-lasting and it was not possible to perform an effective washout. The only transient
PDF effect that occurred in a range of seconds is shown in Fig. 80. In this recording, performed in
saline containing 6 mM CaCl2, all expressed current components (IK, ICa, and Iin,sust) showed a
spontaneous decrease, which was not affected by PDF application (1 fmol, locally applied via
Picospritzer). However, PDF activated an oscillating inward current at +80 mV, most probably a Ca2+
current, which could only be detected in response to the first voltage-step protocol after PDF
application, approximately 15 s after application. In the response to the next protocol, recorded after
45 s, no oscillations were visible. Thus, the effect vanished in less than 45 s (n = 1, Fig. 80).
Tab. 19. Effects of PDF application
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=10
n=2
n=8
n=22
n=4
n=46
21.7 %
4.3 %
17.3 %
47.8 %
8.7 %
INa
n=5
n=2
n=20
n=4
n=4
n=35
14.3 %
5.7 %
57.1 %
11.4 %
11.4 %
ICa
n=9
n=4
n=6
n=8
n=3
n=30
30.0 %
13.3 %
20.0 %
26.7 %
10.0 %
Iin,sust
n=7
n=3
n=9
n=16
n=3
n=38
18.4 %
7.9 %
23.7 %
42.1 %
7.9 %
In 45 whole-cell patch clamp recordings PDF was bath-applied. Effects on different whole-cell current components, such as
the sustained potassium outward current IK, the transient inward currents INa and ICa, and the sustained inward current
Iin,sust were summarized. Percentages relate to the number of cells (# cells) expressing the respective current component.
131
Results
Fig. 77. PDF blocks potassium outward currents as well as sodium and calcium inward currents. A-G. Whole-cell patch
clamp recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage step
protocol: The cell was kept at a holding potential of -60 mV and stimulated with depolarizing voltage steps from -140 mV
to +80 mV to activate voltage-dependent currents. B, C. Representative current traces before (B) and after (C) perfusion
(2 min) with extracellular solution containing the neuropeptide pigment-dispersing factor (PDF, 500 nM) showing a PDFdependent reduction of the sustained outward current (IK, arrow 2), the tail currents (Itail, arrow 3), the first (INa, arrow 4),
and the second transient inward current ( ICa, arrow 5). D. Photograph of the recorded neuron. Scale bar: 20 µm. E-H. I-V
relationships for IK (E, measured at the position indicated by arrow 2 in B), for the transient current components (F, arrow
1), for the mean Itail (G, measured over the range indicated by bar g), and for the negative peak of INa (H, measured over
the range indicated by bar h), measured before (black squares) or after perfusion with PDF (open squares). PDF clearly
reduced the outward current components IK (E) and IK,trans (F) as well as the fast INa (H) and the slower ICa (inward current
component in F). The reduction of the outward currents could also be seen in Itail (G), which was virtually absent after PDF
application. Data for the I-V relationships are given as means ± SEM (n = 3 protocol runs). The data were re-analyzed from
(Yasar 2013).
132
Results
Fig. 78. PDF blocks the current component responsible for the downward bend of the I-V relationship. A-F. Whole-cell
patch clamp recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage
step protocol used to activate voltage-dependent currents. B, C. Representative current traces before (B) and after (C)
perfusion (2 min) with saline containing pigment-dispersing factor (PDF, 500 nM) showing a PDF-independent reduction of
+
the K outward currents (IK, arrow). D. Photograph of the recorded neuron. Scale bar: 20 µm. E. The I-V relationships
(obtained at the position indicated) illustrate the spontaneously decreasing outward current and the PDF-dependent
reduction of a current component responsible for the downward bend at voltages higher than +40 mV, probably mediated
2+
+
by a block of a sustained inward current (I in,sust) or Ca -activated K currents (IK(Ca)). F. The time course of the peak value of
IK at +40 mV (before the downward bend of the current) shows an almost linear reduction, which apparently was not
affected by PDF application (arrow). Data for the I-V relationships are given as means ± SEM (n = 4 protocol runs before,
n = 3 protocol runs after PDF application). The data were re-analyzed from (Yasar 2013).
133
Results
134
Results
+
2+
Fig. 79. PDF blocks K outward currents and Ca inward currents and activates a presumptive Ih current. A-J. Whole-cell
patch clamp recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A. Voltage
step protocol used to activate voltage-dependent currents. B, C. Representative current traces before (B) and after (C)
perfusion (2 min) with saline containing the neuropeptide pigment-dispersing factor (PDF, 500 nM) showing a PDFdependent reduction of the transient outward current (IK,trans, arrow 1), the sustained outward current (IK, arrow 2), the tail
currents (Itail, arrow 3), and the second transient inward current ICa (arrow 5). D. Photograph of the recorded neuron. Scale
bar: 20 µm. E-H. I-V relationships for IK (E, measured at the position indicated by arrow 2 in B), for the transient currents (F,
arrow 1), for the mean Itail (G, measured over the range indicated by bar g), and for the negative peak of the first transient
inward current INa (H, bar h). In the presence of PDF the outward current components of the sustained currents (I K, E, also
shown in (Yasar 2013)), the transient currents (IK,trans, F), and Itail (G) as well as ICa (inward current component in F) were
reduced, while the fast INa (H) was not affected. Data for the I-V relationships are given as means ± SEM (n = 3 protocol
runs). I-K. Next to voltage step protocols, ramp protocols were employed to activate voltage-dependent currents. I. Ramp
protocol, starting with a hyperpolarizing step from Vhold to -140 mV, followed by a permanent increase of the voltage from
-140 mV to +80 mV. J. The corresponding I-V relationships show stronger outward currents as compared to the sustained IK
currents activated by step protocols (E), probably due to superposing hyperpolarization-activated Ih currents. The outward
current decreased PDF-dependently. K. The magnification of the detail marked with the dotted line (J) shows the PDFdependent increase of an inward current, probably via activation of an Ih current. Thereby the reversal potential was shifted
from -52 mV to -46 mV.
135
Results
2+
Fig. 80. PDF causes transient oscillations of a Ca inward current component at +80 mV. A-F. Whole-cell patch clamp
recording from an AME neuron in a primary cell culture (Ø 10 µm, 1 neurite) performed in saline containing 6 mM CaCl2.
A. Voltage step protocol: The cell was kept at a holding potential of -60 mV and stimulated with depolarizing voltage steps
from -140 mV to +80 mV to activate voltage-dependent currents. B, C. Representative current traces corresponding to the
first (B) and the last step protocol of the recording (C). Transient and sustained outward (arrows 1 and 2) and inward
current components (arrows 3 and 4) spontaneously decrease during the recording. D. I-V relationships for all protocol
runs of the recording, calculated at the position indicated by arrow 2 (B), showing a permanent decrease of the sustained
+
K outward current (IK). An inward current component that counteracted IK at voltages higher than +20 mV, resulting in a
downward bend of the I-V curves, also spontaneously decreased. E. The time course for the areas under the I-V curves
(AUC) also illustrates the spontaneous decrease of IK, which was not affected by application of the neuropeptide pigmentdispersing factor (PDF, 100 nM, 1 s, approximately 1 fmol, arrow). F. Magnification of the current traces in response to the
+80 mV voltage step 15 s before PDF application (black), 15 s after PDF application (dark grey), and 45 s after PDF
application (light grey). Only the first current trace after PDF application showed an oscillating inward current
superimposing IK.
3.2.6 Most AME neurons in primary cell cultures are silent
The vast majority of AME neurons in vitro remained silent. Only a few AME neurons (n = 4 of 195,
2.1 %) were detected, which generated spike-like membrane potential changes. Interestingly, all four
recordings were performed with cells without processes in ringer solution containing 6 mM CaCl2. In
one of these recordings an attempt was made to analyze the origin of the events (Fig. 81, Fig. 82).
The events were generated in a regular manner, with similar interevent-intervals, indicated by a
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narrow band in the instantaneous frequency plot and a low coefficient of variance for the interspikeintervals (CV = 0.14, Young et al. 1988; Welsh et al. 1995; Prut and Perlmutter 2003, Fig. 81). The
duration of an event was 4.90 ± 0.17 ms (mean ± SD, n = 10), thus clearly exceeding a regular AP's
duration of 1 - 2 ms (Fig. 35, Fig. 81). While application of TTX and thus block of voltage-dependent
Na+ channels, which drive regular APs, did not affect the events, application of the Ca2+ channel
blocker NiCl2 completely inhibited the activity. Consistently, an increase of [CaCl2]e to 20 mM or full
removal of extracellular Ca2+ also abolished the events, indicating Ca2+ spikes (Fig. 82).
Fig. 81. Single AME neurons may generate spontaneous, regular spikes. A-C. Whole-cell current clamp recording from an
AME neuron in a primary cell culture performed saline containing in 6 mM CaCl2 (same cell as in Fig. 82). A. Recording trace
2+
with a resting membrane potential of -40 mV showing spike-like events, which were most probably Ca spikes (see also
Fig. 82). B. The corresponding instantaneous frequency plot shows a narrow band around 7 Hz indicating regular activity.
C. Magnification of the detail marked with the grey bar above the recording trace (A), illustrating regular intereventintervals.
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2+
Fig. 82. AME neurons may generate Ca spikes. A-G. Patch clamp recording in current clamp mode (cell-attached
configuration) from an AME neuron in a primary cell culture (soma diameter 28 µm, no arborizations) performed in saline
containing 6 mM CaCl2 showing spontaneous activity. The recording traces shown were recorded in the presence of
tetrodotoxin (TTX, 10 nM, A), during perfusion with saline containing 4 mM NiCl2 and 2 mM CaCl2 (B), during washout (C),
2+
during perfusion with saline containing TTX (10 nM) and 20 mM CaCl2 (D), during washout (E), during perfusion with Ca free saline (F), and finally during washout with saline containing 6 mM CaCl2 (G). The spikes were sensitive to NiCl22+
dependent blocking of Ca currents and highly depended on [CaCl2]e while application of TTX alone had no effect,
2+
+
indicating Ca spikes instead of Na -based APs. The increase of the spike amplitude during the recording indicates
spontaneous breaking into the whole-cell mode; probably during washout shown in G. Grey bars above the recording
traces indicate the duration of the respective application.
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3.3 Characterization of peripheral pacemaker neurons of
M. sexta's antennal clock
This chapter focuses on peripheral pacemaker neurons, i.e. pacemaker neurons that are located in a
peripheral tissue and control the function of this tissue. For this purpose olfactory receptor neurons
(ORNs) of the hawkmoth M. sexta were chosen, which most probably control the daytime-dependent
sensitivity of the moth's main olfactory organs, the antennae. The main focus was on the function of
the olfactory receptor coreceptor (ORCO). It was hypothesized that M. sexta ORCO (MsexORCO)
functions as a pacemaker channel that controls SMPOs in the ORNs.
3.3.1 Heterologous expression of olfactory receptors of M. sexta
3.3.1.1 Immunocytochemical characterization of heterologously expressed
MsexORCO
Initially MsexORCO was characterized in a heterologous expression system. Therefore HEK 293 cells
were transiently transfected with MsexOrco and in some experiments cotransfected with the sensory
neuron membrane protein 1 (MsexSnmp-1) and one of the pheromone receptor candidates
MsexOr-1 or MsexOr-4. Before physiological investigations were performed, the expression of the
proteins was examined with immunocytochemistry (Fig. 83). MsexORCO-immunoreactivity was
detected with an antibody against B. mori ORCO (anti-BmorORCO). The anti-BmorORCO antibody
was directed against an epitope of BmorORCO, which identically exists in MsexORCO (Fig. 25).
Expression of the pheromone receptor candidates should be detected with an antibody against
B. mori OR-3 (BmorOR-3), the bombykal receptor of the silkmoth, although the anti-BmorOR-3
antibody epitope differed in six of 16 amino acids of the MsexOR-1 sequence and twelve of 16 amino
acids of the MsexOR-4 sequence (Fig. 26). Since no SNMP-1 antibody was available, it was not
attempted to analyze the MsexSNMP-1 expression.
No MsexORCO-ir cells were detected in control experiments, in which either non-transfected
HEK 293 cells were used (n = 5) or MsexOrco-transfected cells were used but the first (n = 6) or the
secondary antibody was omitted (n = 2). In all cell cultures, which were transfected with MsexOrco
alone, or cotransfected with one of the presumptive pheromone receptors and MsexSnmp-1, a few
cells were clearly MsexORCO-ir (7.0 ± 3.7 %, mean ± SD, n = 35, Fig. 83). Thus, the antibody
specifically detected MsexORCO-immunoreactivity. In contrast to the anti-BmorORCO antibody it
was not possible to detect any signal with the anti-BmorOR-3 antibody, independent of the
transfection profile of the cells (Fig. 83). Neither non-transfected cells (n = 1) nor cell cultures
transfected with MsexOrco only (n = 2) or cotransfected with MsexSnmp-1 and MsexOr-1 (n = 3) or
MsexOr-4 (n = 1) showed any cells expressing BmorOR-3-like immunoreactivity. Thus, it was not
possible to detect the expression of the pheromone receptor candidates MsexOR-1 or MsexOR-4
using immunocytochemistry.
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Fig. 83. Very few transiently transfected HEK 293 cells express MsexORCO. A-I. Maximum projections of confocal image
stacks. HEK 293 cells were transiently transfected with different plasmids and used for immunocytochemistry. Nuclei of the
cells were stained with DAPI (blue). A-C. MsexORCO immunoreactivity (green) was detected with an antiserum directed
against B. mori ORCO (anti-BmorORCO). Only few cells showed MsexORCO immunoreactivity, whether MsexORCO was
expressed alone (A), coexpressed with MsexOR-1 and MsexSNMP-1 (B), or coexpressed with MsexOR-4 and MsexSNMP-1
(C). D-F. The specificity of the MsexORCO immunoreactivity was demonstrated in control experiments using untransfected
cells (D) or using MsexOrco transfected cells but omitting the first antibody (anti-BmorORCO, E), or the secondary antibody
(F). G-I. No immunoreactivity was detected with an antiserum directed against the B. mori bombykal receptor (antiBmorOR-3), whether the cells were transfected with MsexOrco alone (G) or cotransfected with MsexOr-1 and MsexSnmp-1
(H), or MsexOr-4 and MsexSnmp-1 (I). Scale bar: 100 µm.
The expression of MsexORCO was further analyzed with respect to the transfection reagent used for
the transient transfection of the cells (Fig. 84). The percentage of ORCO-ir cells, transfected with
Rotifect transfection reagent (n = 28) was not significantly different from those transfected with
X-tremeGENE transfection reagent (n = 7, p = 0.802, unpaired t-test). Therefore it was not
differentiated between both transfection reagents in all subsequent analyses and the respective
experiments were pooled. Expression of MsexORCO alone (n = 18), coexpression of MsexOR-1
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(n = 7), or coexpression of MsexSNMP-1 and MsexOR-1/4 (n = 10) also did not affect the percentage
of ORCO-ir cells (p = 0.082, one-way ANOVA, Fig. 84, Tab. 28, Tab. 29).
Fig. 84. MsexORCO expression is neither affected by the transfection reagent nor by coexpressed ORs or SNMP-1. A.
Comparison of the percentage of MsexORCO-immunoreactive (MsexORCO-ir) cells, which were transfected using Rotifect
or X-tremeGENE transfection reagent. B. Comparison of the percentage of MsexORCO-ir cells, transfected with MsexOrco
alone, cotransfected with MsexOr-1, or cotransfected with MsexOr-1/4 and MsexSnmp-1 (p = 0.0823). Each data point
represents one cell culture. The lines represent the mean of each data set. Data sets were compared using an unpaired
t-test (A) or a one-way ANOVA (B, n.s. = not significant).
Next, the subcellular localization of MsexORCO was examined. In the majority of ORCO-ir cells, the
signal was detected in large parts of the cell and apparently was not restricted to the membrane
(Fig. 83). Some cell cultures were labeled with Texas Red-conjugated wheat germ agglutinin (WGA),
which selectively binds N-acetylglucosamine and N-acetylneuraminic acid residues in the plasma
membrane, and thus can be used for membrane labeling. On a random basis, some cells, which
showed only marginal ORCO expression, were chosen to generate intensity profiles of the ORCO- and
WGA-fluorescence. Some cells were detected, in which the fluorescence intensity profiles for ORCO
and WGA almost perfectly overlapped, suggesting that MsexORCO could be inserted in the plasma
membrane in these cells (Fig. 85). However, in other cells the intensity profiles only barely
overlapped, suggesting MsexORCO expression apart from the plasma membrane (Fig. 85). Exact
percentages of cells showing presumptive MsexORCO expression in the membrane were not
evaluated. For a further investigation of the subcellular localization of MsexORCO the cells were
subsequently stained with DAPI, which binds to nucleic acids and can be used to label the cells'
nuclei. The DAPI staining revealed that the nucleus of these cells occupied a major part of the cells'
area, while the cytosol occupied only a small region between nucleus and plasma membrane
(Fig. 86). Therefore, ORCO-immunoreactivity in the cytosol and in the plasma membrane could only
hardly be distinguished from each other and the analysis of fluorescence intensity profiles did not
appear to be a reliable method to confirm membrane insertion of MsexORCO.
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Fig. 85. Heterologously expressed MsexORCO is not automatically inserted into the plasma membrane. A. Confocal
image, showing two transiently transfected HEK 293 cells. MsexORCO-immunoreactivity is shown in green, membrane
labeling with Texas Red-conjugated wheat germ agglutinin (WGA) in red. The zigzag lines (b, c) were used to generate the
respective intensity profiles (B and C). B, C. The fluorescence intensities for MsexORCO-immunoreactivity and WGA
measured along the line b (B) largely overlap, while the profiles along the line c (C) barely overlap. Colocalization of
MsexORCO-immunoreactivity and membrane labeling with WGA suggest possible membrane insertion.
Fig. 86. Membrane insertion of heterologously expressed MsexORCO is not unambiguously detectable. A. Confocal
section of transiently transfected HEK 293 cells in a plane with the highest MsexORCO expression. MsexORCOimmunoreactivity is shown in green, membrane labeling with fluorescent WGA in red, and nucleus labeling with DAPI in
blue. Scale bar: 10 µm. B. In the 3D surface plot of the image the fluorescence intensity was plotted in z-direction and
clipped at 50 %. The nucleus occupied the major part of the cells and cytosol and plasma membrane could not be
distinguished reliably.
3.3.1.2 Basic calcium imaging experiments on HEK 293 cells
ORCO orthologues of D. melanogaster (DmelORCO) and other insect species were shown to form
homo- and heteromers with classical ORs and function as Ca2+-permeable cation channels (Sato et al.
2008; Wicher et al. 2008). To investigate, whether MsexORCO functions in a similar way, calcium
imaging experiments, using fura-2 as calcium indicator, were performed.
HEK 293 cells express purinergic P2Y-receptors. These receptors are GPCRs, which respond to
nucleotides, such as ATP, and couple to the IP3 signaling cascade, leading to [Ca2+] rises (Mundell and
Benovic 2000). Therefore, HEK 293 cells were stimulated with different concentrations of ATP
(10-6 - 10-3 M) to control whether the method is working (n = 23). Responsive cells showed an intense
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peak in the fluorescence ratio (F340/F380), indicating [Ca2+] increases. The [Ca2+] increased fast and
declined within 50 - 200 s (Fig. 87, Tab. 30, Tab. 31). The percentage of responsive cells varied from
1 % to 100 % (median: 34.21 %) and apparently increased with increasing ATP concentration,
although not significantly. Higher ATP concentrations (10-4 and 10-3 M) as well as lower ATP
concentrations (10-5 and 10-6 M) activated significantly more cells than control applications of 0.1 %
DMSO.
Fig. 87. HEK 293 cells reliably respond to ATP. A. Normalized calcium imaging data for 42 HEK 293 cells, which were
transiently transfected with MsexOr-1. Each line represents the percentage deviation of the fluorescence ratio from the
-3
mean of the first ten values (% Δ(F340/F380)) for one cell. After application of 10 M ATP (100 µl), 39 of 42 cells responded
with an intense increase of the calcium concentration. B. The percentage of active cells after application of different
-6
-5
concentrations of ATP is shown (n = number of experiments). C. The lower concentrations (10 and 10 M) and the higher
-4
-3
concentrations of ATP (10 and 10 M) were pooled for comparison among each other and with control applications of
0.1 % DMSO (p < 0.0001, Kruskal-Wallis test). Significant differences are indicated by asterisks (n.s. = not significant, *** p
< 0.001; Dunn's multiple comparison test).
Cells heterologously expressing the pheromone receptor candidates were stimulated with lipophilic
pheromone compounds. Since pheromone binding protein (PBP) was not available, an alternative
solvent for the lipophilic compounds was needed. Stengl et al. reported, that PBP could be used
interchangeably with bovine serum albumin (BSA, 10-2 M), which unspecifically binds lipophilic
substances (Stengl et al. 1992b). Control experiments were performed to test, whether HEK 293 cells
respond to BSA, applied at concentrations from 10-5 - 10-3 M. Surprisingly, BSA applications evoked
robust responses (Fig. 88). Compared with ATP applications, the percentage of responding cells
(median: 77.50 %) as well as the strength of the responses was higher for BSA applications. The
percentage of BSA-responsive cells did not differ between non-transfected cells and cells transfected
with MsexOrco and possibly with MsexOr-1 and DmelSnmp-1, indicating that these responses were
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due to the activation of an endogenous receptor of HEK 293 cells (Fig. 88). It was tested, whether the
effects of BSA could be eliminated by the use of essentially fatty acid-free BSA (BSA(-)). Indeed, only a
few cells responded to BSA(-) (median: 4.15 %) and the percentage of responding cells did not
depend on the concentration (Fig. 89, Tab. 30, Tab. 31). While the effects of standard BSA
applications were significantly higher than the effects of the respective control applications and
BSA(-) applications, no difference was detected between BSA(-) applications and the respective
control applications (Fig. 89). Apparently, the prominent effects of standard BSA were mainly due to
the bound fatty acids and BSA(-) appeared suitable to be used as solvent for pheromone compounds.
2+
Fig. 88. BSA elicits intense [Ca ] increases, independent of OR expression. A. Normalized calcium imaging data for 32
non-transfected HEK 293 cells. Each line represents the percentage deviation of the fluorescence ratio from the mean of
-5
the first ten values (% Δ(F340/F380)) for one cell. After application of 10 M bovine serum albumin (BSA, 100 µl), 25 of 32
cells responded with a transient, intense increase of the calcium concentration. B. The percentage of active cells after
application of different concentrations of BSA is shown (n = number of experiments). C. The percentage of active,
untransfected cells was compared with the percentage of transfected cells (n.s. = not significant; Mann-Whitney test;
n = number of experiments).
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Fig. 89. The unspecific responses of HEK 293 cells to BSA are probably due to fatty acids. A. Comparison of the
percentages of active cells after application of essentially fatty acid-free BSA (BSA(-)) in different concentrations (p = 0.909;
Kruskal-Wallis test). B. Comparison of the percentages of active cells after application of BSA containing fatty acids (BSA) or
BSA(-) with the percentages of active cells after application of 0.1 % DMSO. Significant differences are indicated by
asterisks (n.s. = not significant, *** p < 0.001; Mann-Whitney test; n = number of experiments).
Spontaneous [Ca2+] changes were characterized for HEK 293 cells with different transfection profiles
as well as for experiments performed in different bath solutions (Fig. 90, Tab. 30, Tab. 31). Parts of
the experiments were performed in standard ringer solution for HEK 293 cells without supplements,
while other experiments were performed in ringer solution containing BSA(-) or 0.1 % DMSO, which
is also suited to dissolve lipophilic substances. Cells transfected with MsexOrco, MsexOr-1, and
DmelSnmp-1 showed more spontaneous [Ca2+] changes in standard bath solution than nontransfected cells. In bath solution containing DMSO, cells transfected with MsexOrco, MsexSnmp-1,
and MsexOr-1 or MsexOr-4 showed more spontaneous [Ca2+] changes than non-transfected cells. In
contrast, cells transfected with MsexOrco, MsexSnmp-1, and one of the pheromone receptor
candidates were not significantly different from each other, neither in bath solution containing
DMSO nor bath solution containing BSA(-). Likewise, spontaneous [Ca2+] changes of non-transfected
cells and cells transfected with MsexOrco, MsexSnmp-1, and MsexOr-1 did not differ in standard bath
solution compared with bath solution containing DMSO. However, cells transfected with MsexOrco,
MsexSnmp-1, and MsexOr-1/4 showed more spontaneous [Ca2+] changes in bath solution containing
BSA(-) than in standard bath solution or DMSO containing bath solution, although just significant for
the comparison of cells transfected with MsexOrco, MsexSnmp-1, and MsexOr-1 in bath solution
containing DMSO vs. bath solution containing BSA(-). These results suggested that the expression of
MsexORCO and MsexSNMP-1 or DmelSNMP-1 increased the probability of spontaneous [Ca2+] rises.
Since the continuous presence of BSA(-) but not DMSO favored spontaneous [Ca2+] rises, DMSO is
probably better suited as solvent for the pheromone compounds.
The effects of control applications (0.1 % DMSO) in experiments performed in standard bath solution
without supplements were analyzed for cells with different transfection profiles. Non-transfected
cells or cells, transfected with different combinations of MsexOrco, MsexOr-1, and MsexOr-4, were
all significantly less sensitive to control applications than cells, transfected with MsexOrco,
MsexOr-1/4, and DmelSnmp-1 (Fig. 91, Tab. 30, Tab. 31). These results again indicated that the
expression of DmelSNMP-1 increased the activity or the responsiveness of the cells, even to control
applications.
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2+
Fig. 90. Spontaneous [Ca ] changes of HEK 293 cells are increased by the expression of MsexORCO and/or SNMP-1 as
well as the permanent presence of fatty acid-free BSA. A-C. Comparison of the percentages of cells showing spontaneous
2+
[Ca ] changes, either non-transfected or transfected with different combinations of MsexOrco, MsexOr-1, MsexOr-4,
DmelSnmp-1 (DmSnmp), and MsexSnmp-1 (MsSnmp). The underlying experiments were performed in standard
extracellular ringer solution (p = 0.008, Kruskal-Wallis test, A), in ringer solution containing 0.1 % DMSO (p = 0.016,
Kruskal-Wallis test, B), or in ringer solution containing essentially fatty acid-free BSA (C). D-F. Comparison of the effects of
2+
different bath solutions on the percentage of cells showing spontaneous [Ca ] changes. The underlying experiments were
performed with non-transfected cells (D), cells transfected with MsexOrco, MsexOr-1, and MsexSnmp-1 (p = 0.022,
Kruskal-Wallis test, E), or cells transfected with MsexOrco, MsexOr-4, and MsexSnmp-1 (F). For reasons of clarity all
comparisons, not showing significant differences, were omitted in A. Significant differences are indicated by asterisks (n.s.
= not significant, * p < 0.05; A, B, E: Dunn's multiple comparison test, C, D, F: Mann-Whitney test; n = number of
experiments).
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2+
Fig. 91. HEK 293 cells, heterologously expressing DmelSNMP-1, show significantly more [Ca ] increases after control
applications. Comparison of the percentages of active cells after application of 0.1 % DMSO (100 µl, p < 0.0001, KruskalWallis test). The cells either were not transfected or transfected with MsexOrco, MsexOr-1, MsexOr-4, and DmelSnmp-1 in
different combinations. For reasons of clarity only significant differences were indicated (* p < 0.05, ** p < 0.01,
*** p < 0.001; Dunn's multiple comparison test; n = number of experiments).
3.3.1.3 Deorphanization of the pheromone receptor candidates MsexOR-1
and MsexOR-4
Several studies, which were performed in heterologous expression systems and focused on ORCO
orthologues of other insect species, particularly D. melanogaster, B. mori, or A. gambiae (Nakagawa
et al. 2005; Sato et al. 2008; Wicher et al. 2008), demonstrated that ORCO forms heteromeric
complexes with conventional, odorant-binding ORs, in which ORCO (or ORCO homomers) alone or
the heteromeric complex functions as ligand (odorant)-gated non-specific cation channel. To
investigate, whether MsexORCO forms heteromeric complexes with the bombykal receptor, which
mediate bombykal-induced signal transduction, it was necessary to identify the bombykal receptor. It
was suggested that one of the presumptive pheromone receptors MsexOR-1 or MsexOR-4 (malespecific) detects bombykal. The closest relative of MsexOR-1 is the B. mori bombykal receptor
BmorOR-3 and the closest relative of MsexOR-4 is HR13, the H. virescens receptor for the main
pheromone component Z11-hexadecenal (Grosse-Wilde et al. 2010).
Bombykal was dissolved in DMSO or BSA(-) and applied at concentrations ranging from 10-15 to
10-6 M. Stimulation with bombykal did not result in fast, correlated [Ca2+] increases, as expected from
the fast bombykal responses in vivo. The observed [Ca2+] increases after bombykal stimulation highly
varied in their kinetics, with delays and durations ranging from tens of seconds to several minutes
(Fig. 92, Tab. 30, Tab. 31). Since different response kinetics could result from missing signaling
components in the heterologous expression system, kinetics were neglected and any kind of [Ca2+]
increases after the stimulus were detected.
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Fig. 92. HEK 293 cells expressing MsexORCO and MsexOR-1 can respond to high concentrations of BAL with different
kinetics. A-C. Cells were transiently transfected with MsexOrco and MsexOr-1. A. Normalized calcium imaging data for
26 HEK 293 cells. Each line represents the percentage deviation of the fluorescence ratio from the mean of the first ten
-9
2+
values (% Δ(F340/F380)) for one cell. After application of 10 M bombykal (BAL, 100 µl), all cells showed slow [Ca ] increases
-6
with superimposed transient increases. B. BAL (100 µl, 10 M) was applied before the recording started; after more than
2+
200 s 16 of 41 cells showed transient [Ca ] increases. C. The percentages of active cells after application of different
concentrations of BAL or 0.1 % DMSO were compared (p=0.0006, Kruskal-Wallis test, significant differences are indicated
by asterisks). D. Comparison of the percentages of active cells, transfected with MsexOrco and MsexOr-4 (p=0.649,
-12
-9
Kruskal-Wallis test). E. The medial concentrations of BAL (10 and 10 M) were pooled for comparison of cells with
different expression profiles (p = 0.3207, Kruskal-Wallis test). * p < 0.05, ** p < 0.01, *** p < 0.001; Dunn's multiple
comparison test, n = number of experiments.
When cells transfected with MsexOrco and MsexOr-1 were stimulated with different concentrations
of bombykal, only the highest concentration (10-6 M) resulted in a significantly higher percentage of
active cells (median: 6.3 %) compared to the lower concentrations and the solvent control (0.1 %
DMSO). This suggests that MsexOR-1 might be the bombykal receptor (Fig. 92). However, since
micromolar concentration exceed the physiological range, the experiments were focused on lower
concentrations. Cells transfected with MsexOrco and MsexOr-4 were only stimulated with 10-12 and
10-9 M bombykal. As shown for cells transfected with MsexOrco and MsexOr-1, the percentages of
active cells after application of bombykal in these concentrations did not differ from each other or
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control applications. Henceforth experiments employing these concentrations were pooled. The
proportion of responding cells, transfected with MsexOrco and MsexOr-1/4 was not different from
control cells, either non-transfected or transfected with MsexOrco or MsexOr-1 alone (Fig. 92).
In the olfactory system of mice Ca2+-calmodulin (Ca2+-CaM)-modulation plays a key role in ion
channel desensitization and thus response termination and adaptation (Song et al. 2008; Spehr et al.
2009). Therefore experiments were performed to investigate whether the low bombykal-sensitivity
could be due to Ca2+-CaM modulation of the signaling components. Cells transfected with MsexOrco
and MsexOr-1 (n = 4 experiments) or non-transfected cells (n = 5) were incubated for 10 min with the
CaM antagonist W7 before bombykal stimulation (Fig. 93). However, in this experimental series
almost no active cells were detected, neither in control experiments before W7 incubation, nor
during W7 incubation, or after bombykal stimulation.
Fig. 93. The bombykal-sensitivity is not
affected by calmodulin inhibition. Percentages
of active cells after subsequent application of
DMSO, the calmodulin inhibitor W7 (50 µM,
-12
-9
100 µl), and bombykal (BAL, 10 or 10 M,
100 µl) are shown. The transfection profile is
indicated in the legend. Each symbol
represents one experiment.
The low bombykal responsiveness of the cells could have several reasons, including the possibility
that MsexORCO did not function as (part of) an ion channel. To test this hypothesis, one can benefit
from the fact, that ORCO orthologues of different insect species share very high sequence similarities
and may substitute for each other (Jones et al. 2005). First, it was tested, whether a substitution of
MsexORCO by DmelORCO might improve the bombykal sensitivity. Therefore, HEK 293 cells were
transiently transfected with DmelOrco and MsexOr-1 and stimulated with different bombykal
concentrations (n = 3 experiments). Due to the low number of experiments no statistical analysis was
performed, but apparently the percentages of active cells did not differ between different bombykal
concentrations and the solvent control (Fig. 94, Tab. 30, Tab. 31).
Fig. 94. Replacement of MsexORCO by DmelORCO does
not appears to improve the bombykal sensitivity. The
percentages of active cells after application of bombykal in
different concentrations or 0.1 % DMSO are shown. Each
symbol represents the percentage of active cells of one
experiment. The HEK 293 cells were transfected with
DmelOrco and MsexOr-1. Each symbol represents one
experiment.
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Results
Next, MsexOR-1 or MsexOR-4 was heterologously expressed in an insect cell line. For this purpose
SF9 cells were chosen, which previously were shown to express endogenous ORCO (SfruORCO, Smart
et al. 2008). Thus, SF9 cells were transiently transfected with one of the male specific MsexOrs and
stimulated with 10-12 or 10-9 M bombykal. In some experiments [Ca2+] increases after bombykal
application were detected, but the proportion of responding cells was neither different between
MsexOr-1- and MsexOr-4-transfected cells nor between bombykal and control applications (Fig. 95,
Tab. 30, Tab. 31). The expression of endogenous SfruORCO in the SF9 cells was not verified in this
thesis. Since DmelORCO was shown to be activated by cAMP, control experiments were performed
to test the SF9 cells for cAMP sensitivity, which could hint at a possible ORCO expression. In these
experiments slightly more cells responded to the adenylyl cyclase activator forskolin (10-5 M, n = 10),
compared to the solvent control, although not significant (Fig. 127, Tab. 30, Tab. 31).
Fig. 95. SF9 cells, transiently transfected with
MsexOr-1 or MsexOr-4, do not reliably
respond to bombykal. A. Normalized calcium
imaging data for 115 SF9 cells, transfected with
MsexOr-1. Each line represents the percentage
deviation of the fluorescence ratio from the
mean of the first ten values (% Δ(F340/F380)) for
-12
one cell. After application of 10 M bombykal
(BAL, 100 µl, arrow), three cells showed
2+
threshold-exceeding [Ca ] increases with
different kinetics. B. The cells were transfected
-12
with MsexOr-4. After application of 10 M
bombykal three of 124 cells showed threshold2+
exceeding [Ca ] increases. C. The percentages
of active cells after application of bombykal
-12
-9
(10
or 10 M) or 0.1 % DMSO were
compared (n.s. = not significant, MannWhitney test, n = number of experiments). The
transfection profile of the cells is indicated in
the legend.
Another option to exclude MsexORCO as factor of uncertainty was the heterologous coexpression of
the pheromone receptor candidates together with the murine G protein α-subunit Gα15, which was
shown to couple various receptors to the IP3 signaling cascade (Offermanns and Simon 1995). Similar
to the experiments described above, HEK 293 cells, transiently transfected with gα15 and MsexOr-1
(n = 6) or MsexOr-4 (n = 9) did not reliably respond to bombykal at concentrations of 10-12 or 10-9 M.
For each MsexOr-1 or MsexOr-4 some cells showing [Ca2+] increases were detected, but the
percentage of cells was neither different from each other nor from control experiments (Fig. 96,
Tab. 30, Tab. 31). Thus, the low responsiveness to bombykal could neither be improved by
replacement of MsexORCO with DmelORCO or SfruORCO nor by expression of the ORCOindependent Gα15 signaling system.
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Fig. 96. Heterologous expression of the murine
G protein subunit Gα15 does not improve
bombykal-sensitivity. A. Normalized calcium
imaging data for 40 HEK 293 cells, transfected
with gα15 and MsexOr-1. Each line represents
the percentage deviation of the fluorescence
ratio from the mean of the first ten values
(% Δ(F340/F380)) for one cell. After application of
-9
10 M bombykal (BAL, 100 µl, arrow), six cells
2+
showed [Ca ] increases with different kinetics.
B. The cells were transfected with gα15 and
-9
MsexOr-4. After application of 10 M bombykal
2+
one of 105 cells showed a long-lasting [Ca ]
increase. C. The percentages of active cells
-12
-9
after application of bombykal (10 or 10 M)
or 0.1 % DMSO were compared (n.s. = not
significant, Mann-Whitney test, n = number of
experiments). The transfection profile of the
cells is indicated in the legend.
Another possibility for the low responsiveness could be that other crucial parts of the signaling
system, required for pheromone responses, were missing in the heterologous expression systems.
One of those signaling components could be SNMP-1, which is expressed in the pheromone-sensitive
ORNs of M. sexta and other insect species, and is involved in pheromone responses via unknown
mechanisms (Rogers et al. 2001a; Vogt et al. 2009; Li et al. 2014b; Pregitzer et al. 2014). Therefore,
the effects of SNMP-1 coexpression were examined (Fig. 97, Tab. 30, Tab. 31). First, the
D. melanogaster orthologue of snmp-1 (DmelSnmp-1) was used for cotransfection of HEK 293 cells.
Cells, transfected with MsexOrco, MsexOr-1, and DmelSnmp-1, showed more [Ca2+] increases (n = 21,
median: 6.1 %) after bombykal application (10-12 and 10-9 M) than cells, transfected with MsexOrco,
MsexOr-4, and DmelSnmp-1 (n = 17, median: 1.75 %). However, both groups lacked significant
differences with the control groups, which were stimulated with the solvent control. Next, the
M. sexta snmp-1 orthologue (MsexSnmp-1) was cotransfected with MsexOrco and MsexOr-1 or
MsexOr-4, but the dedicated experiments did not show any significant bombykal responses, neither
for those experiments employing bombykal, dissolved in fatty acid-free BSA (compared with the
solvent control), nor for those experiments performed in bath solution containing DMSO or fatty
acid-free BSA (MsexOr-1 and MsexOr-4 compared with each other or spontaneous [Ca2+] increases,
Fig. 97).
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Fig. 97. Coexpression of SNMP-1 does not improve the bombykal-sensitivity. A, B. Percentages of active cells after
-12
-9
application of bombykal (BAL, 10 and 10 M) or the respective control solution containing 0.1 % DMSO (A) or
-6
-4
10 - 10 M bovine serum albumin (BSA, B). The experiments were performed in standard ringer solution. C, D. The
-6
-4
experiments were performed in bath solution containing 0.1 % DMSO (C) and 10 - 10 M BSA (D) respectively. Instead of
2+
applying control solution, spontaneous [Ca ] increases were monitored. HEK 293 cells were transfected with different
combinations of MsexOrco, MsexOr-1, MsexOr-4, MsexSnmp-1 (MsSnmp), or DmelSnmp-1 (DmSnmp). The transfection
profile of the respective cells is shown in the legends. All data are shown as box plots with whiskers (from minimum to
maximum). Data groups were compared using the Kruskal-Wallis test (comparison of the percentages of active cells,
transfected with MsexOrco, MsexOr-1, and MsexSnmp-1 in C, p=0.7147) or Mann-Whitney test (all other comparisons,
A-D). Significant differences are indicated by asterisks (n.s. = not significant, ** p < 0.01, n = number of experiments).
Remarkably, in all experiments performed with cells expressing MsexSNMP-1 or DmelSNMP-1, the
percentage of active cells was slightly higher than in experiments performed without SNMP-1. For
example, the average percentage of active cells, transfected with MsexOrco and MsexOr-1 after
bombykal stimulation (10-12 and 10-9 M) was 0.0 % (Fig. 92 E), while cotransfection of DmelSnmp-1
increased the percentage to 6.1 % (Fig. 97 A, Tab. 30, Tab. 31). However, as shown before (Fig. 90,
Fig. 91) the cotransfection of snmp-1 also increased the percentage of active cells after control
applications as well as the percentage of cells showing spontaneous [Ca2+] increases, explaining the
lack of significant differences in comparisons with the control experiments. Thus, the coexpression of
MsexSNMP-1 or DmelSNMP-1 failed to improve the bombykal responsiveness, but instead elevated
the "general activity and responsiveness" of the cells.
Next to bombykal, the main compound of M. sexta's pheromone blend, one of the minor
compounds, (E,E,Z)-10,12,14-hexadecatrienal, is required for the characteristic, behavioral
pheromone response of the male (Tumlinson et al. 1989). The effect of this compound can be
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Results
simulated by (E,Z)-11,13-pentadecadienal (C-15), which is chemically more stable (Christensen and
Hildebrand 1987). In the following experiments it was tested, whether C-15 might be detected by
one of the male-specific ORs. Thus, HEK 293 cells, transfected with MsexOrco, MsexOr-1/4, and
MsexSnmp-1 were stimulated with low concentrations of C-15 (20*10-15 - 10-12 M). As shown for the
bombykal stimulations almost no responding cells were found, and the rare [Ca2+] increases varied in
their kinetics (Fig. 98, Tab. 30, Tab. 31). Again, the experiments were performed under different
conditions, regarding bath solution and solvent of C-15. While no significant differences were
detected between C-15 stimulation and control experiments for cells expressing MsexOR-1, the
percentage of responding cells, expressing MsexOR-4, (median: 6.58 %) was significantly higher
compared to spontaneous [Ca2+] increases (median: 1.0 %) in experiments, performed in the
continuous presence of 0.1 % DMSO in the bath (Fig. 98).
In conclusion, it was not possible to deorphanize the pheromone receptor candidates MsexOR-1 and
MsexOR-4. The present experiments hint, that MsexOR-1 could be the receptor, detecting bombykal
(Fig. 92, Fig. 97), and MsexOR-4 could be the receptor, detecting (E,E,Z)-10,12,14-hexadecatrienal
(Fig. 98), but an unequivocal identification was not accomplished.
Fig. 98. Cells, heterologously expressing MsexOR-4, can respond to C-15. A. Normalized calcium imaging data for
76 HEK 293 cells transfected with MsexOrco, MsexOr-4, and MsexSnmp-1. Each line represents the percentage deviation of
the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. After application of
2+
(E,Z)-11,13-pentadecadienal (C-15, 100 µl, 20 pM, arrow), five cells showed threshold-exceeding [Ca ] increases with
different kinetics and delays. B. Comparison of the percentages of active cells after application of C-15 (20 fM - 20 pM) or
-5
-6
2+
fatty acid-free bovine serum albumin (BSA(-), 10 - 10 M) and the percentages of cells showing spontaneous [Ca ]
increases respectively (n.s. = not significant, * p < 0.05, Mann-Whitney test, n = number of experiments). The transfection
profile of the cells is indicated in the legend and the bath solution of the underlying experiments at the bottom.
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Results
3.3.1.4 Modulation of heterologously expressed MsexORCO
DmelORCO was shown to function as cation channel that is activated upon odorant stimulation,
when heterologously coexpressed with an OR, conferring odorant sensitivity (Sato et al. 2008;
Wicher et al. 2008). Next to the odorant-gated activation, it was also reported that DmelORCO is
directly activated by PKC and cyclic nucleotides, irrespective if expressed alone or coexpressed with a
classical OR (Wicher et al. 2008; Sargsyan et al. 2011). The experiments described so far aimed at
indirect activation of MsexORCO via pheromone binding to its receptor. In the following experiments
the direct modulation of MsexORCO was investigated. In this set of experiments it was not
differentiated between cells expressing MsexORCO alone or together with MsexOR-1 or MsexOR-4.
The only distinctive feature was the transfection with MsexOrco (orco-positive vs. orco-negative
cells). As in previous experiments, it was not focused on the kinetics and all [Ca2+] increases after
stimulation were equally accounted for.
First, it was tested whether MsexORCO is activated via PKC-dependent phosphorylation. For this
purpose the HEK 293 cells were stimulated with the PKC activator phorbol 12-myristate 13-acetate
(PMA, 10-6 M). The percentages of active cells did not differ between PMA and the solvent control
(0.1 % DMSO) for orco-positive or orco-negative cells, and between PMA stimulation of orco-positive
cells compared with orco-negative cells (Fig. 99). Thus, it was not possible to activate MsexORCO via
PKC-dependent phosphorylation.
Next, the possible cyclic nucleotide sensitivity of MsexORCO was tested. To test cAMP sensitivity, the
adenylyl cyclase activator forskolin and the membrane permeable, hydrolysis resistant cAMP
analogue 8-Br-cAMP were employed. Stimulation of orco-positive cells with forskolin at a
concentration of 40 µM was not different from control applications (n = 8, Fig. 100, Tab. 30, Tab. 31).
2+
Fig. 99. HEK 293 cells, transfected with MsexOrco, do not show significant PMA-dependent [Ca ] increases.
A. Normalized calcium imaging data for 69 cells, transfected with MsexOrco. Each line represents the percentage deviation
of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. Two cells showed threshold2+
exceeding [Ca ] increases after application of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA,
-6
-6
100 µl, 10 M, arrow). B. The percentages of active cells after application of 10 M PMA or 0.1 % DMSO were compared.
Cells with different transfection profiles were pooled and distinguished only by the transfection with MsexOrco (+orco or
-orco; n.s. = not significant, Wilcoxon signed rank test for data pairs (shown in brackets), Mann-Whitney test for unpaired
data, n = number of experiments).
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Results
2+
Fig. 100. HEK 293 cells, transfected with MsexOrco, do not show significant forskolin-dependent [Ca ] increases.
A. Normalized calcium imaging data for 33 cells, transfected with MsexOrco and MsexOr-1. Each line represents the
percentage deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. No cells
responded to the application of forskolin (FSK, 100 µl, 40 µM arrow). B. The percentages of active cells, transfected with
MsexOrco and MsexOr-1 or MsexOr-4, after application of 40 µM FSK or 0.1 % DMSO were compared (n.s. = not significant,
Wilcoxon signed rank test, n = number of experiments).
In contrast to forskolin, stimulation with 8-Br-cAMP at a concentration of 5 µM evoked some [Ca2+]
increases with different kinetics. Slow increases over minutes and fast increases over seconds were
detected (Fig. 101). The percentage of orco-positive cells responding to 8-Br-cAMP was small
(median: 2.35 %, n = 17 experiments) but significantly higher than the percentage responding to
control applications (median: 0.0 %, n = 49) and the percentage of orco-negative cells responding to
8-Br-cAMP (median: 0.0 %, n = 13). No difference was detected between 8-Br-cAMP and control
applications for orco-negative cells. Not every experiment involved control and 8-Br-cAMP
applications. When only those experiments were taken into account (only paired data), 8-Br-cAMP
stimulation did not differ from control applications for orco-positive cells (n = 9) as well as orconegative cells (n = 12, Fig. 101, Tab. 30, Tab. 31).
Then, the sensitivity of MsexORCO to cGMP was tested. Therefore, the cells were stimulated with the
cGMP analogue 8-Br-cGMP (Fig. 102, Tab. 30, Tab. 31). Since no differences were detected for
different concentrations (50 µM or 5 µM), the experiments were pooled for comparison. In the
majority of experiments no clear 8-Br-cGMP effects were detected (median: 0.0 % for orco-positive
(n = 25 experiments) as well as orco-negative cells (n = 13)). Despite equal medians, significantly
more orco-positive cells responded to 8-Br-cGMP compared with control applications. However, no
significant difference was detected between 8-Br-cGMP stimulation of orco-positive and orconegative cells. Only taking into account the experiments involving both 8-Br-cGMP and control
applications (paired data), no significant differences were found for orco-positive (n = 18) or orconegative cells (n = 13, Fig. 102).
Next to the activation via common intracellular signaling molecules such as cyclic nucleotides or PKC,
ORCO activation via specific agonists was also reported. The first substance that was shown to
specifically activate ORCO orthologues of different insect species was VUAA1 (Jones et al. 2011). To
test, whether VUAA1 can activate MsexORCO, HEK 293 cells were stimulated with VUAA1 at a
concentration of 100 µM. The percentage of VUAA1-responsive orco-positive cells was low (median:
3.0 %, n = 71 experiments) but significantly higher than VUAA1-responsive orco-negative cells
(median: 1.0 %, n = 23) or the percentage of orco-positive cells showing spontaneous [Ca2+] increases
(median: 1.0 %, n = 65). Moreover, orco-positive cells showed significantly more spontaneous [Ca2+]
increases than orco-negative cells (median: 0.0 %, n = 30), while no difference was detected between
VUAA1 applications and spontaneous [Ca2+] increases for orco-negative cells (Fig. 103, Tab. 30, Tab.
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Results
31). When only those experiments were considered, where first spontaneous [Ca2+] changes were
measured and then VUAA1 was applied (paired data), the proportion of VUAA1-responsive orcopositive cells was still higher than the proportion of cells showing spontaneous [Ca2+] increases
(n = 43). As shown for other substances, the VUAA1-dependent [Ca2+] increases had different
kinetics. The responses lasted tens of seconds to several minutes and partially were well correlated
with the stimulus (Fig. 103). The present results indicate that VUAA1 is an agonist of MsexORCO, and
that MsexORCO functions as ion channel, which mediates a Ca2+ influx and is spontaneously active.
Fig. 101. HEK 293 cells, transfected with MsexOrco, can respond to 8-Br-cAMP with different kinetics. A. Normalized
calcium imaging data for 62 cells, transfected with MsexOrco. Each line represents the percentage deviation of the
fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. One cell showed a threshold2+
exceeding [Ca ] increase after application of the membrane permeable cAMP analogue 8-Br-cAMP (100 µl, 5 µM, arrow).
2+
B. The cells were transfected with MsexOrco and MsexOr-1; three of 41 cells showed threshold-exceeding [Ca ] increases
after application of 8-Br-cAMP (arrow). C. The percentages of active cells after application of 5 µM 8-Br-cAMP or 0.1 %
DMSO are shown. Cells with different transfection profiles were pooled and distinguished only by the transfection with
MsexOrco (+orco or -orco). D. Only paired data for 8-Br-cAMP and the respective control applications are shown. Data
were compared using the Mann-Whitney test for unpaired data (C, D) and the Wilcoxon signed rank test (D, shown in
brackets) for paired data (n.s. = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, n = number of experiments).
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Results
Fig. 102. Transfection with MsexOrco does not increase the 8-Br-cGMP sensitivity. A. Normalized calcium imaging data
for 30 HEK 293 cells, transfected with MsexOrco and MsexOr-4. Each line represents the percentage deviation of the
fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. None of the cells showed a threshold2+
exceeding [Ca ] increase after application of the membrane permeable cGMP analogue 8-Br-cGMP (100 µl, 50 µM,
arrow). B. The percentages of active cells after application of 5 - 50 µM 8-Br-cGMP are shown. Cells with different
transfection profiles were pooled and distinguished only by the transfection with MsexOrco (+orco or -orco). C. Both
8-Br-cGMP concentrations were pooled. The percentages of active cells after application of 8-Br-cGMP or 0.1 % DMSO
were compared. D. Only paired data for 8-Br-cGMP and the respective control applications are shown. Data were
compared using the Mann-Whitney test for unpaired data (B, C) and the Wilcoxon signed rank test (D, shown in brackets)
for paired data (n.s. = not significant, * p < 0.05, n = number of experiments).
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Results
2+
Fig. 103. Heterologously expressed MsexORCO is activated by VUAA1 and mediates spontaneous [Ca ] increases.
A. Normalized calcium imaging data for 100 HEK 293 cells, transfected with MsexOrco, MsexOr-4, and MsexSnmp-1. Each
line represents the percentage deviation of the fluorescence ratio from the mean of the first ten values
2+
(% Δ(F340/F380)) for one cell. Eight cells showed threshold-exceeding [Ca ] increases with different kinetics after application
of the presumptive ORCO agonist VUAA1 (100 µl, 100 µM, arrow). B. The percentages of active cells after application of
2+
VUAA1 and the percentages of cells, showing spontaneous [Ca ] increases, are shown. Cells with different transfection
profiles were pooled and distinguished only by the transfection with MsexOrco (+orco or -orco). The underlying
experiments were performed in bath solution containing 0.1 % DMSO. C. Only paired data for VUAA1 applications and the
2+
preceding measurements of spontaneous [Ca ] increases are shown. Data were compared using the Mann-Whitney test
for unpaired data (B) and the Wilcoxon signed rank test (C, shown in brackets) for paired data (n.s. = not significant,
* p < 0.05, ** p < 0.01, n = number of experiments). Figure modified after (Nolte et al. 2013).
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Results
In addition to ORCO-activating agents, agents that inhibit heterologously expressed ORCO or
OR/ORCO heteromers have been reported. Among these substances were amiloride derivatives, such
as 5-(N,N-hexamethylene)-amiloride (HMA) and 5-(N-methyl-N-isobutyl)-amiloride (MIA, Pask et al.
2013; Röllecke et al. 2013). Thus, experiments were performed to characterize the effects of those
amiloride derivatives on MsexORCO. When orco-positive cells were stimulated with HMA at a
concentration of 100 µM, all cells showed a clear increase of the F340/F380 ratio (n = 2 experiments),
although one would expect a decrease for the block of a spontaneously active cation channel
(Fig. 104). Closer examination showed, that this effect came about through similar decreases of both
Fig. 104. The amiloride derivatives HMA and MIA elicit unspecific responses, which do not depend on MsexORCO
expression. A. Normalized calcium imaging data for 66 HEK 293 cells, transfected with MsexOrco, MsexOr-4, and
MsexSnmp-1. Each line represents the percentage deviation of the fluorescence ratio from the mean of the first ten values
(% Δ(F340/F380)) for one cell. After application of 5-(N,N-hexamethylene)-amiloride (HMA, 200 µl, 100 µM, arrow) 64 cells
showed threshold-exceeding increases of the fluorescence ratio with similar kinetics. B. Normalized kinetics for 68 nontransfected HEK 293 cells are shown. The bars indicate the application of control solution, 50 µM, or 100 µM HMA via the
perfusion system. All cells showed threshold-exceeding increases of the fluorescence ratio. C. Normalized kinetics for
83 non-transfected HEK 293 cells are shown. The bars indicate the application of ringer solution without supplements
(control) or ringer solution containing 30 µM 5-(N-methyl-N-isobutyl)-amiloride (MIA). 79 of 83 cells showed thresholdexceeding increases of the fluorescence ratio.
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Results
fluorescence intensities, resulting from excitation at 340 nm (F340) and 380 nm (F380, not shown for
HEK 293 cells, see Fig. 112, Fig. 128). Since F340 should increase and F380 should decrease with
increasing [Ca2+] in fura-2-loaded cells, the observed increases of the fluorescence ratio did not
correspond to [Ca2+] increases. To test, whether this phenomenon was ORCO-dependent, nontransfected HEK 293 cells were stimulated with HMA at concentrations of 50 and 100 µM
(n = 3 experiments). Since non-transfected cells responded in exactly the same way to HMA
stimulation, an involvement of ORCO was excluded. In one experiment the effect was shown to
depend on the concentration of HMA (Fig. 104). The second amiloride derivative MIA was only
employed in one experiment performed with non-transfected cells. Since MIA stimulation (30 µM)
produced the same unspecific effect, the experimental series was terminated (Fig. 104).
The next substance under test was N,N-diethyl-m-toluamide (DEET), the world's most commonly
used insect repellent. DEET was suggested to act on the insects' olfactory system and require the
presence of ORCO for its effect (Ditzen et al. 2008). Therefore, it was tested, whether MsexORCO is
inhibited by DEET. DEET was used at a concentration of 100 µM to stimulate non-transfected (n = 1)
or orco-positive cells (n = 8, Fig. 105, Tab. 30, Tab. 31). A few cells showed [Ca2+] increases after DEET
application, but the proportions were not significantly different from control applications (medians:
0.0 %). In none of the experiments prominent [Ca2+] decreases were observed. These results do not
suggest an inhibition of ORCO, but further experiments involving stimulation of ORCO in the
presence of DEET are required to answer the question.
Fig. 105. The presumptive ORCO antagonist DEET does not elicit significant responses. A. Normalized calcium imaging
data for 37 HEK 293 cells, transfected with MsexOrco, MsexOr-4, and MsexSnmp-1. Each line represents the percentage
deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. The bars indicate the
application of bath solution containing 0.1 % DMSO (control), ringer solution containing the presumptive ORCO antagonist
N,N-diethyl-m-toluamide (DEET, 100 µM), and washout with control solution via the perfusion system. Two cells showed
2+
threshold-exceeding [Ca ] increases with different kinetics after application of DEET. B. The percentages of active cells
after application of DEET or 0.1 % DMSO were compared. The cells were transfected with MsexOrco, MsexOr-1/4, and in
some cases with MsexSnmp-1 (n.s. = not significant, Wilcoxon signed rank test for paired data, n = number of experiments).
Finally, the cation channel blocker ruthenium red was employed, which was shown to inhibit
odorant-gated responses of heterologously expressed OR/ORCO complexes of different insect
species as well as VUAA1 induced responses of DmelORCO (Nakagawa et al. 2005; Sato et al. 2008;
Jones et al. 2011; Nichols et al. 2011; Pask et al. 2011; Mukunda et al. 2014). Orco-positive cells were
stimulated with ruthenium red (n = 3). Although no statistical analysis could be performed due to the
low number of experiments, ruthenium red did not seem to inhibit or activate the cells
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Results
(Fig. 106). In contrast to the amiloride derivatives and similar to DEET, ruthenium red apparently did
not evoke unspecific responses and further experiments should be performed to characterize a
possible effect on MsexORCO.
Fig. 106. The presumptive ORCO antagonist ruthenium red does not elicit significant responses. A. Normalized calcium
imaging data for 101 HEK 293 cells, transfected with MsexOrco and MsexOr-4. Each line represents the percentage
deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. The bar indicates the
application of ringer solution containing 100 µM ruthenium red via the perfusion system. None of the cells showed
2+
threshold-exceeding [Ca ] increases. B. The percentages of active cells after application of ruthenium red or 0.1 % DMSO
are shown. Each symbol represents one experiment. The underlying experiments were performed in bath solution
containing 0.1 % DMSO; the cells were transfected with MsexOrco, MsexOr-1/4, and/or MsexSnmp-1.
3.3.2 Characterization of M. sexta ORNs in primary cell cultures
While the experiments described above aimed at a characterization of heterologously expressed
MsexORCO, the following chapter focuses on the function of MsexORCO under less artificial
conditions. Calcium imaging experiments were performed with primary cell cultures, obtained from
dissociated M. sexta antennae. These primary cell cultures contained the presumptive pacemaker
neurons of the antennae, the ORNs, which endogenously express ORCO, allowing for a
characterization in the natural intracellular environment.
3.3.2.1 Fura-2 AM loading of M. sexta ORNs
The ORNs of the primary cell cultures did not show spontaneous activity in the calcium imaging
experiments, suggesting that the uptake of the calcium indicator fura-2 might have been too low for
the detection of weak spontaneous activity. Multidrug resistance transporters (MRTs), such as
multidrug resistance associated proteins (MRPs) and the P-glycoprotein (PGP), belong to the protein
superfamily of ATP-binding cassette transporters, which pump foreign chemical substances
(xenobiotics) out of cell. In X. laevis ORNs and different other cell types MRPs and PGP were
implicated in the active transport of calcium indicators and other fluorescent dyes out of the cells,
which complicates stable calcium imaging experiments and wastes ATP (Manzini and Schild 2003;
Manzini et al. 2008). The first experiments were designed to test, whether loading of the cells with
fura-2 AM could be improved by the use of MRT-blockers. For this purpose the MRP-blocker
probenecid (n = 8 experiments, bath concentration: 2.5 mM) and the PGP-blocker verapamil (n = 3,
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bath concentration: 0.5 mM) were employed. When fura-2 AM loading was performed for 120 min
under optical control and one of the blockers was added after 60 min, the loading progress was
almost linear and no difference before and after the application became obvious (n = 2 experiments
for each blocker, Fig. 107, Tab. 32, Tab. 33), suggesting that fura-2 AM was not transported out of
the cell by MRTs. Spontaneous [Ca2+] increases and [Ca2+] increases after solvent control applications
were not different from cell cultures, in which loading was performed in the absence of probenecid
or verapamil. Thus, the presence of MRT-blockers during incubation with the calcium indicator had
no significant impact on the experiments performed with the respective cell cultures.
Fig. 107. Blockers of multidrug resistance transporters do not affect fura-2 AM loading of M. sexta ORNs, spontaneous
2+
[Ca ] increases, nor responses to control applications. A, B. Normalized loading kinetics of primary M. sexta ORN cell
cultures. Data represent the mean percentage deviation of the fluorescence intensity resulting from excitation at 340 nm
from the mean of the first ten values (% Δ(F340), mean ± SEM). A. Fura-2 AM uptake of n = 39 cells was monitored. The
arrow marks the application of probenecid, an inhibitor of multidrug resistance-associated proteins (10 µl, 250 mM). B. The
effect of verapamil (10 µl, 50 mM, arrow), an P-glycoprotein blocker, on fura-2 AM uptake of n = 35 cells was monitored.
2+
C. The percentages of cells showing spontaneous [Ca ] increases after fura-2 AM loading in ringer solution without
supplements and ringer solution containing probenecid or verapamil were compared (p = 0.7053, Kruskal-Wallis test).
D. The percentages of active cells after control applications (0.1 % DMSO) were compared for different loading
implementations (p = 0.1952, Kruskal-Wallis test).
3.3.2.2 Modulation of ORCO in primary M. sexta ORN cell cultures
The experiments performed on heterologous expression systems indicated that VUAA1 is an
MsexORCO agonist (3.3.1.4, Fig. 103). With this knowledge VUAA1 could be used to characterize the
function of MsexORCO in the ORNs in vitro. When VUAA1 was bath-applied at concentrations of 100,
200, or 500 µM, the majority of cells did not respond with [Ca2+] increases and no concentration
dependence was observed (medians: 0.0 % responding cells for all concentrations, Fig. 108, Tab. 32,
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Tab. 33). Application of the highest concentration (500 µM, n = 12) always failed to activate cells,
although this phenomenon was not statistically significant. No consistent kinetic was detected
among the few [Ca2+] increases after VUAA1 stimulation. In comparison with the solvent control
(0.1 % DMSO) VUAA1 failed to activate a significant higher percentage of cells (n = 28, Fig. 108).
2+
Fig. 108. M. sexta ORNs in primary cell cultures do not show significant VUAA1-dependent [Ca ] increases.
A. Normalized calcium imaging data for 24 presumptive M. sexta ORNs. Each line represents the percentage deviation of
the fluorescence ratio from the mean of the values before stimulation for one cell (% Δ(F340/F380)). The bar indicates the
application of bath solution containing the ORCO agonist VUAA1 (100 µM) via the perfusion system. One cell showed a
2+
threshold-exceeding [Ca ] increase. B. Normalized kinetics of the fluorescence ratio for 21 presumptive M. sexta ORNs.
2+
One cell showed a threshold- exceeding [Ca ] increase after pipette-application of 100 µl VUAA1 (200 µM, arrow), which
already started before the application. C. The percentages of active cells after pipette application of different
concentrations of VUAA1 were compared (p = 0.0720, Kruskal-Wallis test). D. Different VUAA1 concentrations were pooled
and compared with the respective control applications (0.1 % DMSO, n.s. = not significant, Wilcoxon signed rank test for
paired data, n = number of experiments).
Next to VUAA1, other compounds were found to be specific agonists of heterologously expressed
ORCO or OR/ORCO complexes of different insect species (Bohbot and Dickens 2012; Chen and Luetje
2012; Taylor et al. 2012). All of these compounds are structural analogues of VUAA1, which only
differ in minor chemical modifications of the VUAA1 structure (Fig. 30). Interestingly, some chemical
modifications reversed the effect of VUAA1 and resulted in ORCO antagonism (Chen and Luetje 2012;
Jones et al. 2012). Three of these structurally related compounds were tested in the following
experiments, two presumptive agonists (OLC12 and VUAA4) and one presumptive antagonist
(OLC15). When the cells were stimulated with the presumptive ORCO agonists, no prominent [Ca2+]
increases were detected and the percentages of active cells were not significantly higher than for
control applications, neither for OLC12 (200 µM, n = 4 experiments, Fig. 109) nor for VUAA4 (10 µM,
n = 6, Fig. 110, Tab. 32, Tab. 33).
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Similarly, the cells did not respond to stimulation with OLC15 (100 µM, n = 6, Fig. 111, Tab. 32, Tab.
33). Neither the percentage of cells showing [Ca2+] increases (active cells) nor the percentage of cells
showing [Ca2+] decreases (inhibited cells) differed from control applications.
2+
Fig. 109. M. sexta ORNs in primary cell culture do not show OLC12-dependent [Ca ] increases. A. Normalized calcium
imaging data for 20 presumptive M. sexta ORNs. Each line represents the percentage deviation of the fluorescence ratio
from the mean of the values before stimulation for one cell (% Δ(F340/F380)). None of the cells showed a threshold2+
exceeding [Ca ] increase after pipette-application of the ORCO agonist OLC12 (200 µM, 100 µl, arrow). B. The percentages
of active cells after application of OLC12 or 0.1 % DMSO were compared (n.s. = not significant, Wilcoxon signed rank test
for paired data, n = number of experiments).
2+
Fig. 110. M. sexta ORNs in primary cell culture do not show VUAA4-dependent [Ca ] increases. A. Normalized calcium
from the mean of the values before stimulation for one cell (%Δ(F340/F380)). None of the cells showed a threshold-exceeding
2+
[Ca ] increase after application of the ORCO agonist VUAA4 (10 µM, bar). B. The percentages of active cells after
application of VUAA4 or 0.1 % DMSO were compared (n.s. = not significant, Wilcoxon signed rank test for paired data,
n = number of experiments).
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Results
2+
Fig. 111. M. sexta ORNs in primary cell culture do not show OLC15-dependent [Ca ] changes. A. Normalized calcium
from the mean of the values before stimulation for one cell (% Δ(F340/F380)). None of the cells showed a threshold2+
exceeding [Ca ] increase or decrease after application of the ORCO antagonist OLC15 (100 µM, bar). B, C. The percentages
of active cells (B) or inhibited cells (C) after application of OLC15 or 0.1 % DMSO were compared (n.s. = not significant,
Wilcoxon signed rank test for paired data, n = number of experiments).
Finally, the effects of the amiloride derivatives HMA and MIA were tested. Due to unspecific
increases of the F340/F380 ratio, these substances were shown to be unsuitable for the employment in
calcium imaging experiments performed on HEK 293 cells (3.3.1.4, Fig. 104). When HMA or MIA were
applied on primary M. sexta ORN cell cultures, similar unspecific effects were observed: The F340/F380
ratio increased during application to a higher value. During washout the ratio decreased again, often
to a level, which was lower than the starting level (Fig. 112, Fig. 113, Fig. 128). As described before,
the increase of the fluorescence ratio was due to simultaneous decreases of the single fluorescence
intensities for both wavelengths used for excitation of fura-2 (F340 and F380, Fig. 112, Fig. 128). The
percentage of cells showing the F340/F380 increase was higher for 60 µM HMA (n = 6) compared to
30 µM HMA (n = 10), although not statistically significant. Compared to solvent control applications
the percentages of cells showing F340/F380 increases were only significantly different for HMA (n = 16,
Fig. 112) but not for MIA (n = 16, Fig. 113, Tab. 32, Tab. 33). Interestingly, the background
fluorescence was also affected by HMA and MIA application in the calcium imaging experiments.
During application of the amiloride derivatives, the background fluorescence resulting from
excitation at 340 nm remained unchanged, but the background fluorescence resulting from
excitation at 380 nm showed a small increase with the same kinetics like the increase of the cells'
F340/F380 ratio (Fig. 114, Fig. 128). As mentioned before, most cells showed a lower F340/F380 ratio after
washout of the amiloride derivatives compared to the starting level. This phenomenon was due to a
decrease of the F340 intensity and an increase of the F380 intensity, suggesting real [Ca2+] decreases
(Fig. 112, Fig. 128). Consistently, the percentages of inhibited cells were significantly higher for both
HMA (n = 16, Fig. 112) and MIA (n = 16, Fig. 113) compared to control applications.
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Results
Fig. 112. Application of HMA causes cell type-independent, unspecific increases of the fluorescence ratio, resulting from
decreases of the fluorescence intensities of both single wavelengths. A. Normalized calcium imaging data for 23 cells of a
primary M. sexta ORN culture. Each line represents the percentage deviation of the fluorescence ratio (% Δ(F340/F380)) from
the mean of the values before stimulation for one cell. After application of the presumptive ORCO antagonist HMA (30 µM,
bar) 13 cells showed threshold-exceeding increases and five cells showed threshold-exceeding decreases of the
fluorescence ratio. B, C. The normalized fluorescence intensity, resulting from excitation at 340 nm (% Δ(F340), B) or 380 nm
(% Δ(F380), C), shows HMA-dependent decreases for all cells. During washout some cells maintained a lower F340 (B) and a
higher F380 value (C). D. The percentages of active cells after application of HMA at different concentrations were compared
(n.s. = not significant, Mann-Whitney test). E, F. The percentages of active cells (E) or inhibited cells (F) after application of
HMA or 0.1 % DMSO were compared (n.s. = not significant, ** p < 0.01, *** p < 0.001, Wilcoxon signed rank test for paired
data, n = number of experiments).
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Results
Fig. 113. Application of MIA results in cell type-independent, unspecific increases of the fluorescence ratio. A. Normalized calcium imaging data for 23 cells of a primary M. sexta ORN culture. Each line represents the percentage deviation
of the fluorescence ratio (% Δ(F340/F380)) from the mean of the values before stimulation for one cell. After application of
the presumptive ORCO antagonist MIA (30 µM, bar) 14 cells showed threshold-exceeding increases and seven cells
showed threshold-exceeding decreases of the fluorescence ratio. B, C. The percentages of active cells (B) or inhibited cells
(C) after application of HMA or 0.1 % DMSO were compared (n.s. = not significant, *** p < 0.001, Wilcoxon signed rank test
for paired data, n = number of experiments).
In all calcium imaging experiments performed on the presumptive M. sexta ORNs it became obvious
that the cells did not show spontaneous activity manifesting in spontaneous [Ca2+] oscillations. Only
one cell was detected showing robust spontaneous [Ca2+] oscillations. Interestingly, these oscillations
were not affected by application of the solvent control or the MsexORCO agonist VUAA1 but by
application of the amiloride derivatives (Fig. 115). During application of both HMA and MIA the
previously observed unspecific F340/F380 increase occurred and the spontaneous oscillations were
abolished. During washout of the amiloride derivatives the fluorescence ratio decreased, as observed
before. While the spontaneous oscillations returned during washout of HMA, this was not the case
for MIA.
The present experiments suggest that the unspecific increases of the F340/F380 ratio, which were first
observed in HEK cells and then observed in primary ORN cell cultures, were independent of the cell
type. The observed increase of the background fluorescence resulting from excitation at 380 nm was
probably one of the underlying reasons for this artifact. Therefore, the amiloride derivatives HMA
and MIA appeared to be unsuitable not only for experiments performed on HEK 293 cells but for
calcium imaging experiments in general.
169
Results
Fig. 114. The unspecific effects of HMA
on the fluorescence ratio in primary M.
sexta ORN cell cultures may be due to
changes
in
the
background
fluorescence. A. Normalized calcium
imaging data for 16 cells of a primary
M. sexta ORN culture. The data
represent
the
mean
percentage
deviation of the fluorescence ratio
(% Δ(F340/F380), mean ± SEM) from the
mean of the values before stimulation.
During application of the presumptive
ORCO antagonist HMA (60 µM, bar) the
cells showed increases of the
fluorescence ratio. B, C. The background
fluorescence resulting from excitation at
340 nm (% Δ(F340), B) did not show HMAdependent
changes,
while
the
background fluorescence resulting from
excitation at 380 nm (% Δ(F380), C)
showed a mild HMA-dependent increase.
2+
Fig. 115. The amiloride derivatives HMA and MIA block spontaneous [Ca ] oscillations in M. sexta ORNs. Normalized
calcium imaging kinetics for a presumptive M. sexta ORN in a primary cell culture. The percentage deviation of the
fluorescence ratio (% Δ(F340/F380)) from the mean of the first 300 values (first 300 s) is shown. The dotted lines symbolize
breaks in the recording. In the first section of the recording the spontaneous activity of the ORN was recorded. Then, 0.1 %
DMSO, 100 µM VUAA1, 30 µM HMA, and 30 µM MIA were applied via the perfusion system. Application of the amiloride
derivatives HMA and MIA led to the typical, unspecific increase in the fluorescence ratio (arrows) and blocked the
spontaneous oscillations, while the control application and VUAA1 apparently did not affect the activity.
170
Discussion
4 Discussion
4.1 Network analysis of central R. maderae pacemaker
neurons
4.1.1 Network properties of the isolated AME
In chapter 3.1 of the thesis the results of the network analysis of central R. maderae pacemaker
neurons (AME neurons) were reported. Extracellular long-term recordings from the isolated AME
were performed, employing a suction electrode (Schneider and Stengl 2005). Some of the previous
results of the work of Schneider and Stengl such as the regular firing activity and the inhibitory
synaptic interactions were confirmed and some new aspects such as oscillations of the electrical
activity and activating synaptic interactions were found. Schneider and Stengl reported regular
activity in 72 % of the recordings. In the absence of synaptic transmission the activity was increased
(disinhibited). In most of their experiments the cells still fired regularly, but the appearance of new
bands in the instantaneous frequency plot indicated that cells, which were phase-locked before, now
fired with a constant phase difference but still synchronous, causing more than one recurring
interevent-interval. The data suggested that AME neurons were functionally merged in assemblies.
Within one assembly the cells fired regularly with the same or integer multiples of the same
frequency (interevent-interval complies with ultradian period) and with the same phase, while cells
of different assemblies fired with a constant phase difference. The widespread inhibitory synaptic
interactions apparently phase-locked cells of different assemblies (Schneider and Stengl 2005).
4.1.1.1 The AME network mainly contains inhibitory synaptic interactions
Regular firing was detected in 63.1 % of the recordings. Inhibition of synaptic transmission via Ca 2+free saline disinhibited the network activity in 97.9 % of the recordings, and in 52.2 % of these
recordings regular firing still was detected, confirming previous results. However, only one recording
was detected, in which the inhibition of synaptic transmission caused the appearance of a second
instantaneous-frequency band, which indicated a second recurring interevent-interval and thus firing
with a stable phase-difference. The most likely reason for this discrepancy might be that different
neuronal assemblies were recorded. The recording technique implied different sources of variance:
Both kinds of glass capillaries, used as recording electrodes as well as for extirpation of the AME
tissue, were manually broken, resulting in a different size of the AME tissue and thus in a different
number of cells for each recording. The exact position of extirpation slightly varied from experiment
to experiment, causing a different cellular composition of the tissue. Finally, it has not been analyzed
which cells of the tissue (with respect to the position in the suction electrode) contributed to the
recording. Therefore, it seems likely that the recorded cells differed between different experiments.
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Discussion
4.1.1.2 Excitatory synaptic interactions in the AME
Next to the confirmation of the regular activity pattern and the inhibitory synaptic interactions,
excitatory synaptic interactions were also demonstrated. The presence of excitatory chemical
synapses was suggested by recordings showing a decrease of activity or complete quiescence after a
preceding transient activation, when neurotransmitter release was inhibited via Ca2+-free saline.
Since inhibitory neurotransmission was observed in 97.9 % of the respective recordings and
excitatory neurotransmission only in 10.6 %, the AME network appears to contain mainly inhibitory
synapses and only a small proportion of activating synapses. One candidate excitatory
neurotransmitter is acetylcholine (ACh), whose presence in the AME network was demonstrated
indirectly by histochemical detection of the ACh-degrading enzyme acetylcholinesterase in the
glomeruli of the AME (Schendzielorz 2013). Remarkably, in Ca2+ imaging experiments performed with
AME cell cultures nearly all cells (96 %) responded to ACh with [Ca2+] increases and subsequent
oscillations (88 %), mediated via nicotinic ACh-receptors (Baz et al. 2013). At first sight the
widespread ACh-receptivity in the AME contradicts the small proportion of activating synapses,
suggested by the experiments in this thesis. However, ACh was suggested to provide a general
excitatory input into the AME, possibly playing a role in a photic entrainment pathway (Baz et al.
2013). Therefore, it seems likely that ACh-sensitive cells usually were included in the isolated AME
tissue, but the ACh-releasing cells were cut off during the isolation of the AME. If so, ACh-dependent
neurotransmission would be generally in an inactive state in isolated AMAE, and inhibition of
neurotransmitter release usually would not change the activity of the cells receiving ACh-dependent
input.
4.1.1.3 Bursting and oscillations are a characteristic activity pattern of the
isolated AME
Another previously mentioned (Funk 2005) phenomenon was confirmed in this thesis: spontaneous
oscillations or bursting of the AME network occurred in 47.2 % of the recordings with very variable
temporal patterns. This activity pattern included all recordings showing regularly or irregularly
alternating periods of high and low activity and the origin was not further analyzed. It would be
conceivable that synchronized bursting of cells within one or different assemblies contributed to this
activity pattern, probably driven by ultradian SMPOs, which are characteristic for pacemaker neurons
and temporal encoding (Nadasdy 2010; Stengl 2010). Additionally, the contribution of oscillations on
the network level, driven by inhibitory and/or activating synaptic inputs, seems likely. Synaptic
mechanisms might also be involved in the restriction of bursts or oscillations: In 76.6 % of the
recordings, the application of Ca2+-free saline enhanced the occurrence of bursts and oscillations.
However, it cannot be excluded that this phenomenon is Ca2+-dependent and independent of
synaptic transmission. Even if not fully understood so far, this activity pattern appears to play an
important role in the AME network and might also be involved in PDF signaling (see 4.1.3 and 4.2.7).
One should also note that the isolated AMAE used for these experiments missed several important
inputs, which probably resulted in artificial activity patterns. Thus, it is difficult to draw reliable
conclusions referring to the in vivo condition. Preliminary in vivo data collected in extracellular
recordings under my supervision suggested that regular firing as well as bursting are enhanced in the
172
Discussion
isolated AME and occur far less often in vivo (regular firing: 33 %, bursting: 3 % of the recordings,
Brusius 2009). However, this could also mean that regular firing and bursting are endogenous
properties of the AME, which are kept subthreshold or masked in vivo.
4.1.2 Glutamate contributes to inhibitory synaptic interactions of
the AME
It was demonstrated previously, that GABA inhibits the AME network activity in most recordings,
mediated via PTX-sensitive GABAA receptors (Funk 2005; Schneider and Stengl 2005). Since PTX
application simulated the application of Ca2+-free saline, and prominent GABA-immunoreactivity was
detected in the AME (Petri et al. 2002), it was concluded, that the inhibitory synaptic interactions of
the AME are mainly mediated by GABAergic synapses (Schneider and Stengl 2005). Furthermore,
histamine was shown to be an inhibitory neurotransmitter of the AME, since nearly half of the cells in
AME cell cultures responded to histamine in Ca2+ imaging experiments (Baz et al. 2013). Interestingly,
histamine-effects were cimetidin-sensitive (a type 2 histamine receptor antagonist) and suggested to
be mediated via Cl- channels, but could not be blocked by the Cl- channel antagonist PTX (Baz et al.
2013). In this thesis glutamate was identified as another inhibitory neurotransmitter of the AME. The
inhibitory action of glutamate (i.e. the duration of the inhibition) was shown to be dose-dependent,
and the lowest dose eliciting an inhibition was 20 pmol. In comparison with GABA, which was shown
to inhibit AME neurons at doses of 1 - 5 pmol (Schneider and Stengl 2005), the threshold-dose was
higher. Since glutamate-dependent inhibitions were also observed in one recording performed in
Ca2+-free saline, inhibitions were suggested to be direct effects. Glutamate signaling might involve
different types of receptors in insects: In D. melanogaster genes of different (excitatory) ionotropic
glutamate receptor families, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA),
kainate, N-methyl-D-aspartate (NMDA), or δ receptor subtypes have been identified (Littleton and
Ganetzky 2000; Völkner et al. 2000). Additionally, a metabotropic glutamate receptor (DmGluRA,
Parmentier et al. 1996) and a glutamate-gated Cl- channel (GluCl, Cully et al. 1996; Review: Raymond
and Sattelle 2002) were found. Inhibitions of the electrical activity were most probably mediated by
GluCls.
4.1.2.1 Glutamate-dependent inhibitions appeared to be PTX-insensitive
Surprisingly, glutamate-dependent inhibitions were not prevented in the presence of PTX (0.5 mM).
This PTX-insensitivity was only demonstrated in one recording, and only one PTX-concentration was
tested. If the PTX-insensitivity of GluCls can be confirmed in further experiments, bearing in mind
that the inhibitory action of histamine was also shown to be PTX-insensitive, this would suggest, that
PTX-dependent disinhibitions of the AME network activity are indeed mainly due to GABAA-receptor
inhibition. PTX-dependent inhibition of nicotinic ACh receptors, which was reported for example for
A. mellifera AL neurons (Barbara et al. 2005), cannot be excluded for R. maderae AME neurons, but
surely it would not cause a disinhibition. However, inhibitory GluCls were cloned and/or
pharmacologically characterized in different insect species such as D. melanogaster (Cully et al. 1996;
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Discussion
Liu and Wilson 2013), L. migratoria (Janssen et al. 2007), A. mellifera (Barbara et al. 2005), or
P. americana (Raymond et al. 2000; Ikeda et al. 2003; Zhao et al. 2004; Ihara et al. 2005; Zhao et al.
2005; Narahashi et al. 2007; Narahashi et al. 2010). Interestingly, in P. americana dorsal unpaired
median (DUM) neurons as well as A. mellifera AL neurons two types of GluCls are expressed,
mediating a fast, transient (desensitizing) and a slower, sustained (non-desensitizing) current. In
P. americana the sustained but not the transient current is PTX-sensitive (Zhao et al. 2004), while in
A. mellifera the transient current is much more sensitive to PTX than the sustained one (Barbara et
al. 2005). Thus, further experiments are required to analyze, if two types of glutamate-gated chloride
currents can be found in AME neurons of R. maderae, which probably also differ in their PTXsensitivity. Although the PTX-concentration of 0.5 mM was proven to be effective in disinhibiting
the AME network activity (Fig. 44, Schneider and Stengl 2005), higher concentrations might be
employed, since it cannot be excluded that block of GluCls requires higher PTX concentrations than
block of GABAA receptors. Further, the mechanism of PTX-action on GluCls would be interesting. If a
competitive mechanism exists, high doses of glutamate could also be effective in the presence of
PTX. However, the mechanism of PTX-dependent GABAA receptor inhibition suggests a noncompetitive blocking mechanism via interaction with the pore (Newland and Cull-Candy 1992;
Sedelnikova et al. 2006).
4.1.3 Different effects of PDF on the electrical activity of AME
neurons
So far, transient PDF-dependent inhibitions and activations of AME neurons were reported at the
network level, with activations suggested to be indirect effects (Schneider and Stengl 2005). Here,
both classes of effects, activations as well as inhibitions, could be confirmed, but no conclusion about
the directness of the effects could be drawn, since only few recordings in the absence of synaptic
transmission also involved PDF applications (n = 2), and PDF was not effective in these recordings.
Additionally, another class of PDF-effects was observed, PDF-induced bursting or oscillations of the
network activity, which was observed in 20 % of the analyzed recordings involving PDF applications.
As mentioned above, this activity pattern also spontaneously occurred in nearly half of the
recordings (47.2 %) and was induced or enhanced by Ca2+-free saline in 76.6 % of the respective
recordings, suggesting a functional importance for signaling in the AME network. In general, many
synapses act as filters, which do not produce responses to single spikes, but reliably transmit bursts,
since neurotransmitter release is elevated. Thus, bursts may increase the reliability of a synapse.
Considering that the input from one synapse might not be sufficient to exceed the threshold for spike
generation, often coincident inputs and thus coincident bursts from different neurons are required
(Review: Lisman 1997). Consistently, burst synchronization is a common mechanism in neuronal
networks. According to the classical view, a high interspike frequency would always improve synaptic
transmission. Another model includes frequency preferences at the synaptic level (due to short-term
depression and facilitation) and at the neuronal level (due to SMPOs), which might differ between
different postsynaptic cells. Accordingly, synaptic transmission would be highest, when the
presynaptic cell bursts with an interspike frequency, which is resonant for a specific postsynaptic cell.
By changing the interspike frequency different postsynaptic targets with different resonant
frequencies could be addressed (Izhikevich et al. 2003). So far it has not been analyzed, if these
174
Discussion
mechanisms indeed are employed in the AME network, but it seems likely that the observed bursts
and oscillations could play a role in PDF-dependent synchronization or assembly formation
(Schneider and Stengl 2005, 4.2.7).
In the locust L. migratoria PDF was also shown to indirectly increase the firing rate and induce or
strengthen rhythmic activity of motoneurons of the TAG, which was the first electrophysiological
demonstration of a PDF effect in insects (Persson et al. 2001). Such a neuropeptide-dependent
modulation of rhythmic motor patterns is not unusual and best characterized for the stomatogastric
ganglion of crustaceans, where multiple neuropeptides modulate, stabilize, or enhance the rhythmic
pattern (Reviews: Marder and Richards 1999; Marder 2012). However, even if PDF release from AME
neurons is assumed to gate outputs to locomotor control centers (Schneider and Stengl 2005), it is
not clear if the AME network might be comparable with central pattern generating networks, which
directly control locomotor activity.
The PDF effects were shown to be of variable duration (see 3.1.3), including short-term effects of
only some seconds and long-term effects, which lasted for several hours. Interestingly, long-term
effects on the electrical activity, which developed within 30 min and lasted for 2-4 h, were recently
reported for VIP, the mammalian functional homologue of PDF. In contrast to short-term effects
those long-term effects were mediated via clock gene regulation and required higher concentrations
of VIP (Kudo et al. 2013). If the PDF-dependent long-term effects on the AME network were also
mediated via different mechanisms than those mediating short-term effects, has not been analyzed
so far, but would be an interesting topic for future research.
4.1.4 Involvement of cyclic nucleotides in PDF signaling
4.1.4.1 Effects of the cyclic nucleotide analogues were less frequent than PDFeffects
To examine the PDF signal transduction cascade in AME neurons, a possible involvement of the
second messengers cAMP and cGMP was tested. Therefore, the membrane-permeable and
hydrolysis-resistant analogues 8-Br-cAMP and 8-Br-cGMP were applied and the effects were
compared with those of PDF. All classes of PDF-effects could be mimicked by 8-Br-cAMP (Tab. 7), but
not by 8-Br-cGMP. Indeed, there was only one recording, in which the cells started to burst after
8-Br-cGMP application, and in the remaining recordings 8-Br-cGMP did not affect the electrical
activity. Applications of 8-Br-cAMP resulted in fewer effects of each category compared to PDF
applications. This is a surprising result, since both cAMP and cGMP are widespread second
messengers, which should be employed in several signal transduction cascades in a multitude of
different cells. It was expected, that both second messenger analogues affect the electrical activity of
the AME neurons in more recordings than PDF. Probably the small number of cyclic nucleotide
effects indicates an inappropriate application method. Both analogues were delivered in the same
way as PDF: A small volume (nanoliter-range) with a high concentration (millimolar-range) was
applied via pressure ejection (Picospritzer) to the AME-tissue and subsequently slowly washed out. In
many other studies the tissue was incubated in a solution containing the cyclic nucleotide analogues
in lower concentration (micromolar-range) for a longer time (up to 1 h, Prosser and Gillette 1989;
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Discussion
Prosser et al. 1989). Such a long incubation might be more effective when long-term effects are
studied. However, it appears to be useless for the examination of fast, transient effects, as shown for
some PDF applications (Schneider and Stengl 2005). Alternatively, the low number of cyclic
nucleotide effects could indicate the requirement for simultaneous application of other substances
(synergism). Surprisingly, 8-Br-cAMP-dependent effects on the electrical activity occurred with very
short delays in some recordings. In one recording the delay of an 8-Br-cAMP-dependent inhibition
even was below 1 s. In other studies the diffusion of the substance into the cells (C6 glioma cells)
occurred rather slowly, resulting in an uptake of about 6 % of the extracellular concentration during
1 h (Bartsch et al. 2003). In most recordings of this thesis 8-Br-cAMP was employed in a very high
pipette concentration of 10 mM, and often the applications were not performed instantaneously
after the application pipette was located near the AME tissue. Thus, it cannot be excluded that in
some recordings the substance leaked out of the capillary before the application was performed and
documented, thereby causing very small delays. However, in FRET experiments performed with AME
cell cultures the FRET signal increased 8-Br-cAMP-dependently with delays between 10 and 20 s
(Stieger 2011), suggesting that changes of the electrical activity with delays in the range of a few
seconds indeed might be specific effects and not artifacts.
4.1.4.2 cGMP signaling in the AME
Even if the incubation time with the cyclic nucleotide analogues was too short, it is surprising that
8-Br-cGMP affected the electrical activity in only one of 14 recordings, suggesting that the second
messenger cGMP plays only a minor role in the AME. However, there are studies with contrary
results: The levels of cGMP were found to oscillate in the AME (with a minimum at ZT 18) and optic
lobes (with a maximum at ZT 12 in LD and at CT 12 on the first day of DD), suggesting that some
circadian procedures involve cGMP signaling (Schendzielorz et al. 2014). Injections of 8-Br-cGMP into
the complex eye were also effective and shifted the locomotor activity of Madeira cockroaches. The
corresponding phase response curve was of a monophasic all-delay type with a maximum delay at
the beginning of the subjective night. The PRC for 8-Br-cGMP closely resembled the PRC obtained for
myoinhibitory peptide-1 (MIP-1) but not the PDF-PRC, suggesting a function of cGMP in MIP-1
signaling. However, incubation of AME tissue with MIP-1 unexpectedly did not increase cGMP levels
but instead decreased cGMP levels at CT12 (first day in DD, Schendzielorz 2013). Thus, the function
of cGMP in the AME remains unclear, but a contribution in PDF signaling seems unlikely.
4.1.4.3 PDF effects are mediated by cAMP but not cGMP
In D. melanogaster the PDFR belongs to the secretin receptor-like B1 subfamily of GPCRs, which
typically signals via Gαs and Ca2+ (Hyun et al. 2005; Lear et al. 2005b; Mertens et al. 2005). Indeed,
coupling to Gαs was confirmed in different expression systems as well as in situ (Hyun et al. 2005;
Mertens et al. 2005; Shafer et al. 2008; Choi et al. 2012; Duvall and Taghert 2012; Talsma et al. 2012;
Duvall and Taghert 2013; Pírez et al. 2013; Vecsey et al. 2013) and PDF-dependent Ca2+ elevations
were observed in heterologous expression systems (Mertens et al. 2005) and DN1p clock neurons
(Seluzicki et al. 2014). In the experiments of this thesis application of 8-Br-cAMP caused activations,
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Discussion
inhibitions and oscillations of the electrical activity, as shown for PDF. However, 8-Br-cAMP-effects
occurred less frequently than PDF-effects, and only in two recordings similar effects for both PDF and
8-Br-cAMP were demonstrated. In these recordings both substances inhibited the electrical activity
(Fig. 55). These results are no final proof for an involvement of the second messenger cAMP in PDF
signaling in R. maderae but they do suggest that possibility next to other signal transduction
cascades. Even more recordings showing similar effects of PDF and 8-Br-cAMP could not prove Gαssignaling, since it cannot be excluded, that 8-Br-cAMP activates other signaling cascades in AME
neurons, which caused similar effects on the electrical activity. Evidence might be provided by
different antagonists of molecules involved in the putative PDF signal transduction cascade: For
example, reversible attenuation of PDF effects in the presence of AC-inhibitors would indicate an
involvement of AC and PDFR-coupling to Gαs. However, a necessary prerequisite for these
experiments would be the reversibility and the reproducibility of PDF effects, which were not given in
the majority of the experiments. Since electrical activity was recorded at the network-level,
originating from multiple cells of the AME-tissue, additional problems arose. The recorded cells might
have been affected only indirectly by the treatment, and similar effects on the recorded cells could
result from stimulation of different cells and different circuits. Thus, eventually this matter has to be
examined at the level of single cells.
Indeed, the examination of single AME cells was performed in calcium imaging experiments: In one
type of AME cells (type 1) PDF-dependent Ca2+ rises were mimicked by the AC-activator forskolin, and
the effects of PDF as well as forskolin were reversibly blocked by the AC-inhibitor SQ22536 (Wei et al.
2014). Moreover, direct evidence for PDF-dependent elevations of the cAMP level was provided by
El-Sayed Baz and Kai Stieger (University of Kassel) in Förster resonance energy transfer (FRET)
experiments on single AME neurons (Baz 2015). Interestingly, in another type of AME neurons
(type 2) the PDF-dependent Ca2+ increases were not affected by AC-inhibition, suggesting that the
PDFR might also couple to other G proteins than Gαs (Wei et al. 2014). This was also suggested for the
PDFR of D. melanogaster, where an involvement of PLCβ and thus Gαq-signaling in the PDF-dependent
flight control circuit was shown (Agrawal et al. 2013). For further discussion of PDF-signaling see
4.2.7.
4.1.4.4 EPAC-signaling in AME neurons
A few experiments were performed involving the cAMP analogue 8-p-chlorophenylthio-cAMP (007),
which was shown to specifically activate the guanine exchange factor directly activated by cAMP
(EPAC). In its activated state EPAC catalyses the GDP-to-GTP-exchange of the monomeric G proteins
RAP1 and RAP2, and thus their activation (Enserink et al. 2002; Review: Gloerich and Bos 2010).
Interestingly, the firing rate was irreversibly inhibited by 007 in one third of the recordings in
standard saline (n = 2 of 6) and in all recordings in Ca2+-free saline (n = 2), suggesting that inhibitions
are direct effects, suggesting a higher efficiency than 8-Br-cAMP, and a contribution of EPAC in
cAMP-dependent inhibitions of the firing rate in AME neurons. This is a surprising result, since 007
should not activate any cAMP targets, which are not activated by the less specific 8-Br-cAMP.
However, 007 was employed at pipette concentrations of 10 mM, and in contrast to 8-Br-cAMP it is
highly lipophilic, suggesting a better membrane permeation (Bartsch et al. 2003). Although the
activation of type-1 or type-2 PKA holoenzymes required hundredfold higher concentrations of 007
177
Discussion
than the activation of EPAC (Enserink et al. 2002), unspecific effects on other cAMP targets such as
PKA and CNG or HCN ion channels cannot be excluded at a pipette concentration of 10 mM. Thus, in
future experiments the expression of EPAC in AME neurons should be identified first and subsequent
biochemical experiments should be performed to test the specificity of different cyclic nucleotide
analogues.
Recently, both EPAC and PKA were reported to be involved in long-lasting effects of VIP on SCN
neurons (Kudo et al. 2013), since the effects were prevented by antagonists of each molecule.
However, none of the recordings involving applications of 007 also involved PDF application. Thus,
the experiments of this thesis do not allow a speculation about an involvement of EPAC in PDF
signaling.
neurons in primary cell cultures
4.2.1 Primary cell cultures of AME neurons
So far the electrophysiological characterization of R. maderae AME neurons has been limited to
extracellular recordings from the isolated AME (Schneider and Stengl 2005, 2006, 2007) and a few
intracellular recordings, aimed to characterize light-sensitive optic lobe neurons (Loesel and
Homberg 1998; Loesel and Homberg 2001). In order to obtain information about electrophysiological
properties of single AME pacemaker neurons, primary AME cell cultures were employed for patch
clamp experiments. These AME cell cultures consisted of different neuronal and non-neuronal cell
types of different size and morphology and were characterized by a low survival rate, resulting in loss
of most cells during the first two weeks in vitro. These cell cultures were originally developed by Petri
and Stengl (1999) and described as long-term cell cultures, which survived for more than two months
in vitro, and consisted of at least six morphological cell types. Collecting hemocytes from hemolymph
of cockroach larvae and adding these cells to the AME cell cultures (hemocyte conditioning) did not
affect survival of the cells but increased the percentage of cells with processes from approximately
20 % to 40 % and improved the outgrowth of processes, which reached a length of up to 600 µm,
apparently due to growth factors released by the hemocytes (Petri and Stengl 1999). Since hemocyte
conditioning was not reported to improve the survival rate of the cells, it was omitted at the cost of
non-optimal outgrowth of neurites. Different attempts were made to improve the survival rate,
including the use of different cell culture protocols, variations in the enzymes for dissociation, in the
composition of the media, in the incubation conditions, as well as in the age and condition of the
cockroaches that were sacrificed for cell culture preparation. All these strategies failed to improve
the survival rate of the cells in vitro. The quality of the cell cultures, described by Petri and Stengl
(1999), could not be achieved, most probably due to a change of some parameters, whose
significance has not been recognized to date. Therefore, the short life-span of the cell cultures was
accepted and patch clamp experiments were restricted to the first few days in vitro.
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Discussion
It can be assumed that an AME neuron inevitably undergoes physiological changes, when it is
removed from its cellular context, the optic lobes of the brain, to culture conditions in a different
chemical milieu. During preparation and dissociation it loses its complete neurites, its synaptic input
from other neurons, as well as influences from other cell types such as glial cells. One could argue
that the cell's physiological state in culture progressively moves away from its natural state, and the
sooner an experiment is performed the closer the artificial state is to the natural state. Since adult
cockroaches were used to prepare cell cultures, the AME neurons should be fully differentiated, and
there should not be any requirement for a developmental period in culture. From this point of view a
restriction of experiments to the first few days in vitro absolutely makes sense. However, with
respect to the damage, caused by the tissue dissociation, it seems appropriate to use the cell cultures
not until a longer period of recovery, in which the neurons may regenerate for example their
processes, which are without doubt a critical part of a neuron. Thus, further attempts should be
made to achieve long-term cultures of a higher quality, as described before (Petri and Stengl 1999).
In any case, the effects of hemocyte conditioning should be tested, even if it was not reported to
enhance the survival rate. In preliminary experiments hemocytes were successfully collected and
kept in culture (not shown).
As mentioned before, the AME cell cultures consisted of different cell types. This is not surprising,
since no selection mechanisms for a specific cell type were employed. AME explants were previously
described to contain 1400 ± 260 somata (Petri and Stengl 1999) or 970 ± 258 somata (mean ± SD,
Reischig and Stengl 2003a). The differing cell numbers indicate that the number of cells highly
depends on the size of the glass capillary, which is used to excise the tissue, as well as on the precise
location of the excision. The AME explants contain all adjacent neurons that send processes to the
AME (250 - 300 neurons), except for a group of neurons located in the posterior cell cortex (Petri et
al. 1995; Reischig and Stengl 1996; Petri and Stengl 1999). Thus, the percentage of AME-associated
pacemaker neurons is approximately 25 %. About 90 % of the neurons were reported to get lost
during the dispersion procedure, yielding 10 % of the neurons of the excised tissue, which may settle
down (Petri and Stengl 1999). Thus, only an unknown proportion of the cells, which can be found in
the AME cell cultures, are pacemaker neurons, which associate with the AME in vivo. Unfortunately,
there were no morphological criteria to identify these pacemaker neurons in vitro. For the patch
clamp experiments cells with arborizations were favored, since it was considered that arborizations
may identify neurons, and that only cells in a good condition may regenerate their neurites.
However, AME-associated pacemaker neurons could not be identified by their arborizations. Thus, it
can be assumed, that only some neurons chosen for the experiments were pacemaker neurons. Any
kind of marker, which could help to identify specific living AME neurons, would be very helpful.
Unfortunately, no such marker is available at the moment. Fluorescence markers, driven by
promoters of specific genes, would be a useful tool. Since AME cells are best characterized by their
expression pattern of specific neuropeptides or neurotransmitters, the corresponding genes would
be suitable targets (Petri et al. 1995; Reischig and Stengl 2003b; Schulze et al. 2012; Review:
Homberg et al. 2003). Less knowledge is available for the expression pattern of specific clock genes
involved in the molecular feedback loop of the pacemaker neurons. Preliminary immunocytochemical experiments showed a widespread expression pattern of PER, which is unsuitable for
the identification of specific neurons, and the expression pattern of other clock genes has not been
characterized so far (Werckenthin 2013). However, no genetically modified Madeira cockroaches
have been developed until now, and thus, no genetically encoded fluorescence markers are
available. Another possibility for the identification of a specific subset of AME neurons could be the
179
Discussion
injection of fluorescence-coupled neuronal tracer molecules, such as dextrans. If injected into
specific tracts, these tracers will be transported into the corresponding somata of the neurons. In this
way, specific neuronal subsets could be marked, for example AME-associated GABA-ir neurons via
injections into the distal tract (Petri et al. 2002) or a subset of the PDF-ir neurons as well as other
neurons via injections into the commissures, which connect the brain hemispheres (Reischig and
Stengl 2002; Reischig et al. 2004). However, given the enormous loss of 90 % of neurons during tissue
dissociation, it is questionable whether an AME cell culture, prepared after injection, would contain
any fluorescence-marked neurons at all. Thus, the injection method seems not to be very efficient,
leaving only the identification of neurons by physiological characteristics at the moment.
4.2.2 AME neurons express different whole-cell current components
Although many cells died in the effort to establish a whole-cell patch clamp recording, several
recordings could eventually be performed. Mean resting membrane potentials (VRMP) of -35.59 mV
(6 mM Ca2+) or -37.75 mV (1 mM Ca2+) were measured (Tab. 8), indicating that the neurons were in a
depolarized state, which probably correlated with the low survival rate of the cell cultures. Different
current components and oscillations were detected and characterized (Tab. 9). Oscillations persisted
in the presence of TTX (Fig. 72) but not NiCl2 (Fig. 73). In one recording PDF-dependent oscillations
solely occurred at +80 mV (Fig. 80). Thus, the phenomenon was caused by oscillating Ca2+ currents,
possibly via CaM-dependent mechanisms. The high proportion of cells expressing Ca2+ currents
(65 %) indicates that these currents were not blocked by concanavalin A, a lectin originally extracted
from the jack-bean Canavalia ensiformis, which was used to coat the coverslips for the cell culture
dishes. Such an inhibition has been reported before (Ivens and Deitmer 1986; Lucas and Shimahara
2002).
All current components were detected in both experimental series (performed in saline containing
6 mM or 1 mM Ca2+) and the different Ca2+ concentrations only slightly affected the abundance of the
single current components. The abundance of Itail showed the largest difference between both
experimental series (13 %). Certainly, the different manifestations of Iin,sust and activationcharacteristics of the transient inward currents INa and ICa differed depending on the Ca2+
concentration. However, the second experimental series was not performed at the same time as the
first series, and the recordings as well as preparations of the corresponding AME cell cultures were
performed by other researchers at that time. Thus, different important factors changed between
both experimental series and the observed differences might not solely be due to the different Ca2+
concentrations employed.
Interestingly, the amplitude or even the occurrence of different current components was highly
influenced by the stimulation protocol. When cells were stimulated with depolarizing voltage steps
from a holding potential of -60 mV, the smallest values for IK were detected, and in approximately
65 % of the recordings the transient ICa was present. Additionally, most recordings showed the high
voltage-activated Iin,sust, which counteracted IK. Stimulation with voltage ramps clearly increased IK
(Fig. 79), and stimulation with step protocols including a preceding hyperpolarization (-140 mV) also
boosted IK and prevented activation of the transient ICa (Fig. 63). Likewise, Iin,sust was absent, highly
reduced, or shifted to higher voltages (Fig. 64). The shift to higher voltages was only detected by
180
Discussion
chance, and step protocols covering a voltage range up to +120 or +140 mV are required to
investigate it. Most likely, ICa and Iin,sust were not blocked by the hyperpolarization, but instead
masked by the superimposed, increased outward currents. Moreover, a preceding hyperpolarization
activated sustained inward currents at voltages more negative than Vrev and rendered the I-V
relationship for the sustained outward currents towards a linear increase, suggesting the activation
of non-specific cation currents. A candidate current, which is activated by hyperpolarization, is the
current flowing through HCN channels (Ih, Hille 2001). The expression of this current in AME neurons
was previously demonstrated (Wei and Stengl 2012; Wei et al. 2014). Whether Ih solely contributed
to the increased inward currents at negative voltages or additionally increased the outward currents
at positive voltages, remains to be investigated. Likewise, the permeability to Ca2+ remains to be
analyzed, which was reported to be very low in other species (Review: Craven and Zagotta 2006).
Putative Ca2+ influx through HCN channels might activate IK(Ca), which could contribute to the
increased outward currents. Alternatively, increased outward currents after a preceding
hyperpolarization might be caused by the activation of A-type K+ currents (IA). However, these
currents were reported to be transient, inactivating currents (Grolleau and Lapied 1995; Hille 2001;
Wicher et al. 2001), while the hyperpolarizing prepulses activated non-inactivating outward currents
in AME neurons.
4.2.3 Ion channels underlying IK and Iin,sust
In approximately 95 % of the recordings the voltage-dependent increase of IK stopped around
+30 mV or +40 mV, where it reached a plateau or decreased again, sometimes resulting in an I-V
relationship showing an "N-shape" (Fig. 59). This phenomenon was termed Iin,sust. It could mean, that
IK indeed first increased until a specific voltage and then decreased (Heyer and Lux 1976).
Alternatively it could be interpreted as a sustained inward current that activated at higher voltages
and counteracted IK to a different extent. Obviously, different current components contributed to
this phenomenon. An interplay of a pure voltage-dependent, delayed rectifier-type IKdr and a Ca2+activated K+ current (IK(Ca)) as reported by Lucas and Shimahara (2002) is suitable for explanation. In
that study IKdr was shown to have an I-V relationship showing a simple rising function and IK(Ca) to
have a bell-shaped I-V relationship (Fig. 116, Lucas and Shimahara 2002). When different proportions
of both currents are superimposed, I-V relationships showing a plateau, a downward bend, or the
characteristic "N-shape" might result. However, in approximately 50 % of the recordings, IK not only
slightly decreased, but instead the downward-bend of the I-V curves reached prominent negative
values (Fig. 64). These intense non-inactivating inward currents sometimes had higher absolute
values than the sustained outward currents of the corresponding cells. They cannot be explained as
IK(Ca) since the dependence on Ca2+ cannot cause a K+ outward current to become an inward current
at voltages more positive than +40 mV. Considering the extracellular and intracellular ringer
solutions, IK should theoretically reverse at -93.2 mV from an inward current to an outward current.
In recordings showing a strong Iin,sust the non-inactivating currents turned negative at voltages around
+50 mV. This indicates the activation of a new sustained Ca2+ inward current since Ca2+ currents
theoretically have reversal potentials of +154.6 mV (6 mM Ca2+) or +132.0 mV (1 mM Ca2+). In some
recordings Iin,sust was activated in the presence of TTX. Thus, an underlying sustained Na+ current can
be excluded (see 4.2.5). Thus, both components, IK(Ca) and sustained Ca2+ currents, might counteract
181
Discussion
the pure voltage-dependent IKdr and contribute to the plateau or the decrease of the sustained
current. Since it could not be clearly differentiated between these components, the sustained
outward current was termed IK, irrespective of a contribution of IKdr or different IK(Ca) components. All
components counteracting IK at higher voltages, resulting in the plateau or the downward bend of
the I-V curves were termed Iin,sust, irrespective of a contribution of different IK(Ca) or sustained Ca2+
inward currents. In this way, the outward current and the counteracting components could be
analyzed and evaluated separately from each other. As a consequence, specific effects on IK(Ca) were
not evaluated, since interference with IK(Ca) should affect the amplitude of the outward current as
well as its decrease at higher voltages (respectively its counteracting components). Thus, it is too
difficult to differentiate from interference with IKdr or sustained Ca2+ inward currents without highly
specific pharmacological treatment.
Fig. 116. Superposition of IKdr and IK(Ca) might result in an N-shaped I-V relationship. A-C. I-V curves obtained in a wholecell patch clamp recording of an olfactory receptor neuron (ORN) of M. brassicae. A. Before treatment the I-V relationship
2+
was N-shaped (open circles), indicating that IK(Ca) as well as IKdr were included. Blocking of Ca currents and hence IK(Ca) with
2+
CoCl2 resulted in the solely voltage-dependent I-V curve for IKdr (filled circles). B. Subtraction of the Co -resistant current
2+
2+
from the Co -sensitive current revealed the bell-shaped I-V curve for IK(Ca). C. Probably due to a rundown of Ca currents,
the I-V relationship developed from the N-shape (open circles) towards a simple rising function (filled circles) during a
recording. Redrawn and modified after (Lucas and Shimahara 2002).
4.2.4 All current components spontaneously decrease during wholecell recordings
Most of the cells died some minutes after establishment of the whole-cell configuration,
accompanied by a darkening or shrinkage of the cell. While a decrease of the brightness and loss of
the halo, which are both characteristics of healthy cells, indicates a deterioration of the cell's
condition, shrinkage may indicate a loss of volume probably due to the osmolarity of the recording
solution. Since other cells of the respective culture, which were not used for a recording, did not
shrink, the osmolarity of the extracellular solution did not appear to be responsible. Furthermore,
extracellular solution of the same composition was used for the extracellular recordings of the
isolated AME (see 3.1), which usually lasted for several hours. In rare cases, these recordings allowed
for measurement of electrical activity up to 86 hours (Funk 2005), indicating that the extracellular
ringer solution is quite appropriate for AME neurons. Therefore, it was tested whether the problem
can be solved with an adjustment of the pipette solution's osmolarity, which usually was adjusted to
lower values than the bath solution. However, adjustment to the same or higher values than the bath
solution did not prevent the problem.
182
Discussion
When recordings with durations of at least 4 min and without pharmacological treatment of the cells
were analyzed, it was found that usually all whole-cell current components showed a spontaneous
decrease (Tab. 10). The phenomenon of a spontaneous reduction of different currents recorded in
the whole-cell configuration and particularly in cell-free patches was reported in several publications
and termed rundown. Rundown was detected for HVA L-type, N-type, P/Q-type, and R-type VACCs,
but not for LVA T-type VACCs. Next to these Ca2+ channels it was reported for other ion channel
families such as K+ channels (ATP-inhibited K+ channels (K(ATP)), inward and outward rectifier K+
channels (Kir and Kor), Ca2+-activated K+ channels (K(Ca)), Na+-activated K+ channels (K(Na)), epithelial K+
channels), different Cl- channels (e.g. GABAA receptors), Na+ channels, and N-methyl-D-aspartate
(NMDA) receptors (Reviewed in Becq 1996; Hille 2001). Rundown of Ca2+ channels, particularly the
cardiac L-type channel, is best studied. It is neither due to an artificial artifact nor to a lack of
membrane integrity. The amplitude of single channel currents is not decreased during rundown.
Instead the open probability of single channels or the number of active channels in a patch is
reduced. Hence, the reduction is not based on proteolysis of ion channels, but due to washout of
important cytoplasmic factors by the artificial solution (Becq 1996; Kepplinger and Romanin 2005).
Addition of high energy compounds such as Mg-ATP (Kostyuk et al. 1981; Belles et al. 1988; Ono and
Fozzard 1992; Kameyama et al. 1997; Hao et al. 1999), the catalytic subunit of PKA, different
phosphatase inhibitors (Kostyuk et al. 1981; Armstrong and Eckert 1987; Ono and Fozzard 1992), and
calpastatin (an inhibitor of the protease calpain, Romanin et al. 1991; Seydl et al. 1995; Kameyama et
al. 1998; Hao et al. 1999) partially could prevent rundown. Thus, phosphorylation of the respective
ion channels, direct ATP-binding, as well as an unknown function of calpastatin support the
prolonged activity. In addition, rundown is promoted by elevations in [CaCl2]i, mediated by the
endogenous Ca2+ sensor CaM (Peterson et al. 1999; Qin et al. 1999; Review: Halling et al. 2006), and
the activity of the channels might be stabilized by binding of phosphatidylinositol 4,5-bisphosphate
(PIP2, Hilgemann and Ball 1996; Huang et al. 1998; Wu et al. 2002; Gamper et al. 2004). For the AME
neurons used in the experiments of this thesis, it cannot be excluded that the spontaneous decrease
of different current components is due to a lack of membrane integrity, since many cells died during
recordings, and often all current components of a cell simultaneously decreased. No experiments
involving excised patches were performed to investigate this phenomenon at the level of single
channel currents. However, it seems likely that the observed current reductions are at least partially
due to rundown. Prominent Ca2+ currents could activate Ca2+ dependent processes, such as CaMdependent promotion of rundown. Different Ca2+ currents most probably were subject to rundown.
In turn this could promote rundown of other ion channels, such as K(Ca) channels (Lucas and
Shimahara 2002), which apparently contributed to the whole-cell current. Furthermore, AME cells
could also express K(Na) channels, as reported for DUM neurons of P. americana (Grolleau and Lapied
1994; Wicher et al. 2001). In rat olfactory bulb neurons KNa channels were shown to require
intracellular Na+ concentrations of 10 - 180 mM for activation and to exhibit a rundown (Egan et al.
1992). Since no Na+ was included in the pipette solution, current components showing a Na+dependence such as the presumptive IK(Na), should inactivate, when the pipette solution elutes the
cytosol. To investigate the origin of the spontaneous current decrease several experiments should be
performed in the future: The decrease should be investigated at the level of single channel currents
in excised patches. The perforated-patch technique should be employed, where access to the interior
of the cell is not established by rupture of the membrane within the patch pipette, but instead by
antibiotic-dependent permeabilization, which prevents washout of macromolecules (Marty and
Finkelstein 1975; Lindau and Fernandez 1986; Horn and Marty 1988; Rae et al. 1991; Kyrozis and
Reichling 1995). Furthermore the pipette solution should include Na+ and Mg-ATP, since Na+183
Discussion
dependent currents cannot be excluded and Mg2+ as well as ATP might play a role in rundownattenuation. In addition, the effects of other substances, implicated in the prevention of a rundown
could be tested, such as phosphatase- and CaM-inhibitors, as well as calpastatin and the regulatory
subunit of PKA. On the other hand one could test, whether the currents remain stable when the
recording conditions are mitigated. For example, the duration of the stimulation protocols could be
reduced by 50 %, since no changes in the second half of the current responses were detected.
4.2.5 Pharmacological characterization of the current components
For the pharmacological characterization of the observed whole-cell current components different
ion channel blockers were employed. Due to the instability of the recorded cells, it was usually not
possible to test different substances in one recording. Accordingly, control applications and washout
of the substances could not be performed in the same recordings. The spontaneous decrease of the
current components complicated the analysis of these experiments. When one or several current
components decreased following the application of an ion channel blocker, the reduction could not
solely be attributed to the respective blocker. Therefore, the area under the I-V curves for a
particular current (or alternatively the peak current) was plotted against the recording time to obtain
the time course for the current decrease. Current changes were only attributed to the blockers, if
this time course showed a correlation with the application. By the use of this method a qualitative
analysis of the pharmacological experiments was possible, but no quantitative conclusions could be
drawn.
The ion channel blockers employed in this study usually affected several current components, most
probably some of them directly and others indirectly (see 3.2.4). First, the contribution of different K+
channels was analyzed by the use of TEA, 4-AP, apamin (applied from outside), and CsCl (applied
from inside). TEA, applied from outside, was supposed to inhibit different K+ currents, such as IKdr, IA,
IKir, IK(ATP), and big conductance K(Ca) currents (IBK, Hille 2001). As expected, IK and IK,trans were reduced
in more than 80 % of the recordings (Tab. 12, Tab. 25). The TEA-dependent increase of INa might be
explained as indirect effect due to the loss of counteracting outward currents. Inhibition of ICa
indicates a connection between IK and ICa, which also became obvious in the recordings employing
CsCl. The TEA-dependent decrease of Iin,sust suggests block of IK(Ca), probably BK channels, and an
increased Iin,sust might be caused by a change of [CaCl2]i.
CsCl, applied from outside, should block all K+ channels, while it should only block IKdr, when applied
from inside, as it was performed in this thesis (Hille 2001). However, other publications report a
broader spectrum of K+ target channels, when applied from inside (Hewes 1999). In all recordings
employing CsCl the outward currents were reduced, but never completely blocked (Fig. 69). Even in
the presence of CsCl and TEA residual outward currents (Iout,res) were present. Since TEA alone
attenuated IK only partially, and CsCl probably blocks only IKdr from inside, Iout,res could indicate an
incomplete block of K+ currents. SK channels for example should neither be blocked by TEA nor by
CsCl in the patch pipette (Hille 2001). Furthermore, outward currents in the presence of CsCl and TEA
have been reported to be carried by Cs+ ions flowing through Ca2+ channels (Byerly and Hagiwara
1982; Fenwick et al. 1982). Most probably non-specific cation currents and outward rectifying
Cl- currents also contributed to the residual outward current. This is also supported by GABA184
Discussion
applications, which provoked an almost linear, outward rectifying current, apparently due to Cl- influx
(Fig. 76). This current was suggested to be a Cl- current, since GABA was shown to act predominantly
via GABAA-receptors in AME neurons (Schneider and Stengl 2005). However, GABA was applied only
in two recordings, and the resulting currents did not resemble typical GABA-gated Cl- currents,
measured in DUM neurons of P. americana (Alix et al. 2002; Zhao et al. 2003, 2004). In future
experiments this should be pharmacologically investigated, for example by the use of the GABAAreceptor antagonist PTX, which is very effective on AME neurons (Schneider and Stengl 2005).
Another characteristic of the Cs+-dependent block of K+ currents was a change in the inward current
composition. The second transient inward current ICa, which activated around +50 mV in standard
ringer solution and was also blocked by TEA, was never detected in the presence of CsCl. Instead,
another Ca2+ current was detected, which activated around -50 mV, peaked at -10 mV, comprised an
inactivating and a non-inactivating component, and masked the considerably smaller INa (Fig. 69,
Fig. 74). Obviously, this Ca2+ current was superimposed by Cs+-sensitive K+ currents in standard ringer
solution, where it counteracted IK and contributed to its high reversal potential (Vrev, Tab. 8).
However, the calculated Vrev of -93.2 mV for IK suggests that mainly other non-identified inward
currents, which activate at lower voltages, are responsible for the measured values of Vrev. In future
experiments the block of K+ channels could be intensified by application of both CsCl and TEA via the
pipette solution and the bath solution, since TEA also works from inside and Cs+ also works from
outside (Hille 2001).
The membrane-permeable 4-AP blocks the sustained IKdr as well as the transient IA (Hille 2001).
Applied on AME neurons, 4-AP was not as effective as TEA or CsCl (Tab. 13, Tab. 26). Blocking of IK
and IK,trans were expected, while blocking of INa, ICa, and Iin,sust might be unspecific or indirect effects.
The smaller number of cells responding to 4-AP reflects its higher specificity compared to TEA and
CsCl and suggests that IKdr and IA only played minor roles in the neurons, which were exposed to 4-AP.
In this case, the main components of the sustained outward current might have been IK(Ca), IK(Na), or ICl,
next to non-specific cation currents. The transient outward current IK,trans usually showed only a weak
expression (Fig. 60, Fig. 63), not comparable to the strong A-type currents measured in honeybee
Kenyon cells (Schäfer et al. 1994) or cells expressing the D. melanogaster genes shaker or shal
(Review: Wicher et al. 2001). Moreover, both IK(Ca) and IK(Na) may have transient, fast inactivating
components, as shown for neurons of P. americana (Grolleau and Lapied 1994; David and Pitman
1995; Grolleau and Lapied 1995) or A. mellifera (Schäfer et al. 1994; Cayre et al. 1998; Review:
Wicher et al. 2001). Thus, IK,trans does not necessarily need to be an A-type current, but could also
consist of fast IK(Ca) and IK(Na) components. In contrast, the strong, hyperpolarization-dependent
increase of outward currents suggested the presence of IA, although not only inactivating but also
non-inactivating outward currents were increased (Fig. 63, Fig. 64).
To begin the pharmacological characterization of K(Ca) channels, which possibly contributed to IK and
Iin,sust and could explain the observed rundown of IK, the honey bee venom apamin was employed,
which should specifically block SK channels (Hille 2001). Apamin-dependent reductions of IK, Iin,sust,
and Itail suggested a contribution of SK channels to these current components in some AME cells
(Tab. 14). The reduction of ICa in some cells was observed for all K+ channel blockers and might always
be an indirect effect, which in turn could also affect the Ca2+-dependent Iin,sust. In Ca2+ imaging
experiments performed on primary AME cell cultures apamin increased the baseline and the
frequency of spontaneous Ca2+ transients and deceased the amplitude of the transients, suggesting a
function of SK channels in repolarization and restraining of the activity (Wei and Stengl 2012).
185
Discussion
However, in other studies performed on insect neurons neither apamin nor other SK antagonist were
shown to be effective (French 1989; Grolleau and Lapied 1995; Mills and Pitman 1999; Peron and
Gabbiani 2009), although genes encoding for SK channels were detected (Review: Wicher et al.
2001). For a detailed analysis of K(Ca) channels, further experiments employing apamin are required.
Additionally, specific antagonists for BK channels or intermediate-conductance K(Ca) channels (IK) as
well as different K(Ca)-agonists could be tested (Hille 2001; Peron and Gabbiani 2009). Furthermore,
the general contribution of IK(Ca) might be examined indirectly via the block of Ca2+ currents or by the
use of Ca2+-free saline.
Ca2+ currents were further characterized by the use of NiCl2 and mibefradil. Divalent ions like Cd2+,
Co2+, Mn2+, or Ni2+ can block different Ca2+ channels with different blocking efficiency, which can be
used to differentiate between Ca2+ channel types (Hille 2001). Here, only NiCl2 was employed
(Tab. 16, Tab. 17). The high proportion of cells showing a block of ICa or ICa(Cs) confirmed that these
currents were indeed Ca2+ currents, and the high proportion of cells showing an IK block suggested
that a main part of the outward current is IK(Ca). However, the residual outward current Iout,res in the
presence of CsCl was only partially blocked by NiCl2, indicating other outward current components
next to the Cs+-sensitive K+ current and IK(Ca), as suggested before. The block of INa again might be an
unspecific effect and the block of Iin,sust is a logical consequence of a direct block of Ca2+ channels and
the consequent indirect block of IK(Ca). However, in 5 of 10 recordings under standard conditions NiCl2
increased the Ca2+-dependent Iin,sust, suggesting that the Ca2+ homeostasis of these cells was in a
different state, and the block of Ca2+ currents shifted the Ca2+ homeostasis in a direction that allowed
for Iin,sust. In further experiments a contribution of Ca2+ release from internal stores to this
phenomenon could be investigated (Reviews: Srikanth and Gwack 2012; Hooper et al. 2014).
The second Ca2+ channel blocker employed was mibefradil, which was initially considered to be a
specific LVA VACC antagonist (Osterrieder and Holck 1989), but then found to be a non-selective
antagonist that blocks all subclasses of Ca2+ channels (Bezprozvanny and Tsien 1995; Viana et al.
1997). Since mibefradil blocked the complete Ba2+ current in embryonic neurons of P. americana, it is
also effective in insects (Benquet et al. 2000; Benquet et al. 2002). All actions of mibefradil on AME
neurons (Tab. 18) might be explained in the same way as for NiCl2, although mibefradil was less
effective (i.e. less cells were affected). In Ca2+ imaging experiments on AME neurons mibefradil
reduced the baseline level of [Ca2+]i and blocked spontaneous Ca2+ transients in almost all AME
neurons (Wei and Stengl 2012). It was suggested, that mibefradil blocked LVA VACCs, which
functioned as pacemaker channels facilitating spontaneous activity. In the patch clamp experiments
of this thesis neither widespread spontaneous activity nor widespread LVA inward currents were
detected. Although the high Vrev for IK suggested counteracting inward currents at negative voltages
around the calculated K+ equilibrium potential, only Ca2+ currents activating around -50 mV were
detected when K+ channels were Cs+-dependently blocked. Under standard conditions Ca2+ channels
even activated at positive voltages. Small, transient LVA inward currents only rarely were observed in
single recordings (Fig. 60, Fig. 69). However, given the high VRMP of the AME neurons beyond -40 mV,
even a Ca2+ current activating at -50 mV could provide a small amount of inward current at VRMP.
Non-specific cation currents through cyclic nucleotide gated (CNG) or hyperpolarization-activated
cyclic nucleotide-gated (HCN) channels could also act as pacemaker channels and provide an inward
current at hyperpolarized potentials (Hille 2001; Craven and Zagotta 2006). Since the HCN channel
antagonist DK-AH269 was reported to decrease the Ca2+ baseline level and block Ca2+ transients in
64 % of spontaneously active AME neurons in Ca2+ imaging experiments (Wei and Stengl 2012), a
186
Discussion
proportion of AME neurons should express these channels. When voltage ramps or step protocols
including hyperpolarizing pre-pulses were employed for stimulation (Fig. 64, Fig. 79), a possible
contribution of cation currents was detected (see also 4.2.2). This was not the case, when voltage
step protocols starting from the holding potential were employed, indicating that non-specific cation
currents did not activate or were masked by other current components. A superposition of nonspecific cation currents and IK at negative voltages might also explain the high reversal potential of IK
between -40 mV and -60 mV. A pharmacological characterization of HCN and CNG channels as well as
a possible modulation of whole-cell current components by cyclic nucleotides was left aside for
future investigations.
Another possibility for a pacemaker current would be a sustained Na+ current as shown for rat SCN
neurons (Kononenko et al. 2004). Non-inactivating or slowly inactivating Na+ currents were also
described for some insect neurons, such as P. americana DUM neurons (Lapied et al. 1990),
A. mellifera Kenyon cells (Schäfer et al. 1994), and D. melanogaster embryonic neurons (O'Dowd
1995; Review: Wicher et al. 2001). No sustained Na+ currents have been detected in this study and in
Ca2+ imaging experiments only 11 % of AME neurons showed small Ca2+ baseline reductions, when
TTX was applied, suggesting that sustained Na+ currents only play a minor role in these neurons
(Wei and Stengl 2012). TTX is a potent and specific blocker of sustained and transient Na+ currents
(Hille 2001). Consistently, TTX reduced the fast INa in 100 % of the patch clamp recordings. Other
current components were less frequently affected (Tab. 15). The reduction of IK could be explained
as indirect effect on a presumptive IK(Na), which is activated by Na+ influx, as shown for P. americana
DUM neurons (Grolleau and Lapied 1994). The probably unspecific decrease of ICa might explain the
TTX-dependent reductions of IK (due to an inhibition of IK(Ca)) and activations of the Ca2+-dependent
Iin,sust. In future experiments Na+ currents could be further analyzed by replacing Na+ ions in the
recording solutions with ions that may not pass the pore of the Na+ channels, such as choline.
A small set of recordings with control applications was performed. In one of these recordings the
current components IK, INa, and ICa decreased after the application of pure bath solution (not shown).
This suggests that some of the pharmacological experiments might have had unspecific effects,
which were not due to the respective substance. In future experiments the endurance of the cells
has to be improved to increase the recording duration, which in turn would facilitate control
applications, washout, and repetitive pharmacological stimulation in one recording.
4.2.6 Most neurons in AME cell cultures do not spike
Surprisingly, only a small proportion of approximately 2 % of the recorded AME neurons (n = 4
of 195) spontaneously generated spike-like events, although 70 % of the neurons expressed voltagegated Na+ currents. Consistently, resting membrane potentials more positive than -40 mV indicated a
depolarized state of the neurons, which probably prevented spiking (Fig. 58). Missing spontaneous
activity could also indicate a lack of pacemaker current expression, which activates at hyperpolarized
potentials and drives the cell to spike threshold. In general, cells with neurites, where voltage-gated
Na+ channels are supposed to be expressed, were favored for patch clamp recordings. However,
none of the four spiking cells had regenerated its neurites and all of them were recorded in the first
experimental series in saline containing 6 mM CaCl2.
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Discussion
The low number of spontaneously spiking cells contrasts the observations that have been made in
extracellular recordings of isolated AMAE (see 3.1, Schneider and Stengl 2005, 2006, 2007) and
calcium imaging experiments performed on primary AME cell cultures (Wei and Stengl 2012).
Extracellular recordings from isolated AMAE always showed events, which were considered as
summed action potentials (SAPs), originating from the superposition of APs generated by different
neurons at the same time. The AMAE were excised in the same way like the AMAE used to prepare
primary cell cultures, and the recordings were performed in the same ringer solution as used in the
first experimental series of patch clamp recordings (6 mM CaCl2). However, in the extracellular
recordings network activity of an unknown number of neurons was recorded, and the proportion of
spontaneously active pacemaker neurons was not examined. Thus, there is a chance that the activity
originated from a small proportion of pacemaker neurons that drove the activity of otherwise silent
neurons. Blocking of synaptic transmission was reported to increase the network activity, indicating
widespread inhibitory synaptic interactions in the network, such as GABA-ergic ones (Petri et al.
2002; Schneider and Stengl 2005, 2006). If the network is driven by a few pacemaker neurons,
blocking of synaptic transmission should rather inhibit than activate the network activity, but
inhibitions were only rarely detected (see 3.1.1). Next to chemical synapses, gap junctions appear to
play a role in the AME (Schneider and Stengl 2006). Thus, even in the absence of chemical synaptic
transmission some pacemaker neurons may drive the activity of other neurons. Consistently, the
network activity was shown to cease after application of the gap junction blocker halothane
(Schneider and Stengl 2006).
In calcium imaging experiments performed on the same primary AME cell cultures 26 % of the cells
showed spontaneous, rapid Ca2+ transients, which were TTX-sensitive (Wei and Stengl 2012). It was
concluded that, the rapid Ca2+ transients were triggered by TTX-sensitive APs. Since TTX only rarely
affected the Ca2+ baseline concentration of the cells, the effect was apparently not due to a block of
sustained Na+ pacemaker channels but to a block of voltage-gated Na+ channels involved in the
generation of APs (Wei and Stengl 2012). While 10 nM TTX were sufficient for a complete block of
the electrical activity of AME explants (Schneider and Stengl 2005, 2007), this concentration only
reduced amplitude and frequency of the rapid Ca2+ transients in AME cell cultures, where the single
cells are by far more easily accessible. Furthermore, 10 nM TTX also blocked the fast transient ICa in
the patch clamp experiments and increased the non-inactivating inward current Iin,sust in each 30 % of
the recordings, demonstrating that TTX effects are not restricted to Na+ channels in these neurons.
Thus, it seems that there is no direct evidence for APs in the imaging studies, even if the sensitivity to
TTX suggests underlying APs. On the other hand, the low survival rate of the cells in vitro as well as
the spontaneous decrease of several whole-cell current components indicates that the AME cell
cultures were not in an optimal condition. Considering that a whole-cell recording is a more serious
interference for the cell than an imaging experiment, it seems likely that the condition of the cells in
imaging experiments was slightly better than the condition after breaking through the membrane in
whole-cell patch clamp experiments. Therefore, a higher number of spiking cells in imaging
experiments might have been due to a better condition of the cells. Another reason could be the
loading of the cells with the calcium-sensitive dye fura-2, which actually is the only difference
between the cells used for imaging and patch clamp experiments. It was shown that BAPTA-based
calcium sensitive dyes might induce non-intrinsic phenomena: fluo-4 for example induced Ca2+ spikes
in rat SCN cells (Hong et al. 2010). For that reason, further experiments might be performed, in which
calcium imaging and patch clamp are employed simultaneously. Alternatively, cells loaded with
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Discussion
fura-2 and treated in the same way as in calcium imaging experiments may be used for further patch
clamp experiments, to test for unspecific effects of the high affinity calcium dye.
In one of four AME somata showing spontaneous spike-like events, these events could not be
identified as TTX-sensitive, Na+ based APs, but instead as prolonged Ca2+ spikes. The Ca2+-dependence
was suggested, since the activity of the neuron was not affected by 10 mM TTX alone, but attenuated
by 10 nM TTX in combination with an elevated Ca2+ concentration (20 mM), by Ca2+-free solution, or
NiCl2. However, this characteristic was only observed in one recording and insufficient concentration
of TTX cannot be excluded. Interestingly, isolated ventromedial cells of M. sexta, which secrete
eclosion hormone, were also shown to spike, although their processes were lost (Hewes 1999). Ca2+
spikes with long durations have also been described before, for example for neurosecretory cells
(NSCs) of the silkmoth B. mori. In contrast to APs generated by non-neurosecretory cells, those APs
had a duration of 39 ± 11 ms and were only dependent on Ca2+ in the soma but Ca2+ and Na+ in the
axon near to the soma (Miyazaki 1980). The long duration was probably caused by the weak
expression of the voltage-dependent IKdr. The Ca2+ influx into the soma was suggested to play a role in
exocytotic secretion of neurohormones from the soma or in the initiation of axonal transport
(Miyazaki 1980). Prolonged, overshooting APs seem to be a general characteristic of APs in insect
neurosecretory cells and have been reported for neurosecretory cells of B. mori (Miyazaki 1980),
P. americana (Cook and Milligan 1972; Wicher et al. 1994), or M. sexta (Hewes 1999). Although the
exact cellular identity of the cells in primary AME cell cultures is unknown (see 4.2.1), the multitude
of peptidergic neurons associated with the AME (see 1.4.5) suggests, that these cell cultures
contained different neurosecretory cells. Thus, it is likely, that the spontaneously active neuron
(Fig. 81, Fig. 82) was a neurosecretory cell. Consistently with a neurosecretory function, strong
transient Ca2+ currents were detected in approximately 65 % of the AME neurons. These currents
might be required for Ca2+-dependent neuropeptide release, oscillations, and spikes.
4.2.7 Effects of PDF on single AME neurons
Finally, the effects of the neuropeptide PDF on whole-cell current components of AME neurons were
investigated. Apparently PDF is the most important coupling signal of the circadian clocks of fruit flies
and cockroaches, being absolutely required for behavioral rhythmicity in DD and synchronized
activity of different clock neurons (Schneider and Stengl 2005; Lee et al. 2009; Review: HelfrichFörster 2014). While much is known about PDF-effects on behavioral and molecular oscillations, it
was only begun to examine the signal transduction cascade(s). So far there is only little insight in
acute PDF-effects on the electrical activity of clock neurons and the participating ion channels, which
were examined in this part of the thesis.
All current components under investigation were affected by PDF, and in most recordings reduced
(Tab. 19). Only when voltage ramp protocols were used for stimulation, PDF-dependent activation of
putative non-specific cation currents (Ih) was observed, resulting in an inward current at
hyperpolarized potentials (Fig. 79). Additionally, PDF activated an oscillating Ca2+ current at +80 mV
in one recording (Fig. 80). Special care was taken to exclude spontaneous current changes, which
were not correlated with the application. However, some application-dependent but nevertheless
unspecific current changes cannot be excluded, since such artificial effects were demonstrated for
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Discussion
one control application and the limited recording duration did not allow for subsequent applications
of PDF and control solution in the same recording (see 4.2.5).
The multitude of target ion channels was not expected, but might be explained by different
signalosome compositions: In D. melanogaster the PDFR couples to Gαs (Hyun et al. 2005; Mertens et
al. 2005; Shafer et al. 2008; Choi et al. 2012; Duvall and Taghert 2012; Talsma et al. 2012; Duvall and
Taghert 2013; Pírez et al. 2013; Vecsey et al. 2013) and probably also to Gαq (Agrawal et al. 2013). In
R. maderae multiple lines of evidence also suggest Gαs coupling (see 4.1.4.3) and at least in one type
of AME neurons (type 2) PDF signaling does not employ AC (Wei et al. 2014). If the PDFR might
couple to different G proteins in one cell, as shown for the heterologously expressed adipokinetic
hormone receptor of P. americana (Wicher et al. 2006), has not been examined so far. PDF signaling
in D. melanogaster remarkably requires highly specific signalosomes, which include different
A-kinase anchor proteins (AKAPs) and differ in distinct clock cell types (Duvall and Taghert 2012,
2013). The involvement of PKA was not only suggested by the presence of AKAPs but also
demonstrated for PDF effects on the molecular feedback loop in DN1p clock neurons (Seluzicki et al.
2014). If one assumes, that PDF signal transduction is similar in the fruit fly and the Madeira
cockroach, it seems likely that the R. maderae PDFR is also a component of cell type specific
signalosomes. If the cAMP level is PDF-dependently increased, different signaling pathways are
possible. Possible targets of cAMP are ion channels, such as non-specific HCN or CNG cation channels
(Review: Craven and Zagotta 2006) or ether-à-go-go-type K+ channels (Brüggemann et al. 1993;
Delgado et al. 1995). Furthermore, PKA and EPAC could be activated, each of both molecules having
another set of target molecules. EPAC for example might be involved in the regulation of Ca2+induced Ca2+ release (CICR) from the endoplasmatic reticulum and could mediate a crosstalk
between cAMP and PLC/PKC-signaling (Review: Gloerich and Bos 2010). Of course PKA also has a
multitude of possible target molecules; ion channels are of particular importance in this context: Na+
channels might be inhibited (Li et al. 1992; Cantrell et al. 1997; Catterall 1999) and Ca2+ channels
might be activated by PKA-dependent phosphorylation (Wicher 2001). Likewise, phosphorylation by
PKC modulates ion channel function (Numann et al. 1991; Catterall 1999; Wicher 2001). Probably all
ion channels can be modulated by phosphorylation, often by different protein kinases, and
dephosphorylation (Reviews: Levitan 1994; Wicher et al. 2001). Thus, depending on the composition
of signalosomes and the expression of ion channels in close vicinity PDF could target several ion
channel types. If the PDFR would couple to more than one G protein in the same cell, or if G protein
switching was employed (Daaka et al. 1997; Krsmanovic et al. 2003), accordingly more ion channels
could be affected.
In studies performed on D. melanogaster constant activation of PDF-autoreceptors or acute
activation of misexpressed PDFR in motoneurons resulted in depolarizations (Choi et al. 2012; Vecsey
et al. 2013). During the preparation of this thesis a study was published showing PDF-dependent Ca2+
increases in clock neurons of D. melanogaster (DN1p, see 1.3.3). Furthermore, signaling via cAMP
was confirmed and an involvement of PKA was demonstrated for PDF-dependent resetting of the
molecular feedback loop, whereas its depolarizing effect on the DN1ps' membrane potential was
mediated via cAMP-dependent but PKA-independent activation of an LVA inward current, probably a
non-specific cation current (Seluzicki et al. 2014). PDF-dependent Ca2+ increases were also observed
for AME neurons in Ca2+ imaging experiments (Wei et al. 2014; Baz 2015). While some AME neurons
showed Ca2+ increases (type 1 and 2 neurons), other AME neurons (type 3 and 4) responded with a
decrease of [Ca2+]i (Wei et al. 2014).
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Discussion
A current similar to the presumptive cation current observed by Seluzicki et al. was also activated by
PDF in the experiments of this thesis, even though the kinetics were different. Seluzicki et al. could
demonstrate, that the current activates within few seconds after PDF application. Here, the current
changes usually were detected after 2 - 5 min. In both studies the currents were long-lasting but the
exact duration was not analyzed. The effects often persisted and grew stronger over minutes.
However, Seluzicki et al. locally applied PDF (50 µM) for 10 s and constantly perfused the recording
chamber, while in most experiments of this thesis PDF (500 nM) was slowly washed in (2 min) via the
perfusion system, and except for these 2 min the recording chamber was not perfused. The observed
effects of slowly applied PDF suggest that the steepness of the PDF concentration changes was not
crucial for the PDFR of R. maderae. However, given the different kinetics and the fact that the only
transient PDF effect was detected after local application of PDF (100 nM) for 1 s, the application
parameters might be improved in future experiments. Likewise, the effects of constant perfusion and
thus washout of PDF could be examined.
Substantially more knowledge is available in mammals for VIP, the functional homolog of PDF. VIP
and another peptide, PACAP, both activate the same receptors with different affinity. The receptor
PAC has a high affinity for PACAP, while the VPAC1 and VPAC2 receptors have the same affinity for
VIP and PACAP (Harmar et al. 1998). Only PAC1 and VPAC2 are expressed in SCN neurons (Usdin et al.
1994; Sheward et al. 1995; Cagampang et al. 1998). The PAC1 receptor couples to Gαs and Gαq/11
(Harmar et al. 1998) and VPAC receptors were also shown to signal via cAMP and PKA (Rea 1990;
Vanecek and Watanabe 1998; Meyer-Spasche and Piggins 2004; An et al. 2011) as well as PLC (An et
al. 2011; Harmar et al. 2012; Kudo et al. 2013). Activating as well as inhibitory effects of VIP on
different ion channels in different cell types have been demonstrated in several studies: Non-specific
cation currents and particularly Ih currents were activated (Wang and Aghajanian 1989, 1990; Sun et
al. 2003; Hermes et al. 2009). However, in another study performed on SCN slices, HCN channels
were not affected by VIP (Atkinson et al. 2011). Different HVA VACCs (L-, N-, and P/Q-type), but not
LVA T-type VACCs were inhibited (Ehrlich and Elmslie 1995; Zhu and Yakel 1997; Hayashi et al. 1999;
Hayashi et al. 2002). Different potassium currents were activated, such as an SK-type IK(Ca) (Haug and
Storm 2000) and the fast delayed rectifier current (IFDR), which mediates fast repolarization (Kudo et
al. 2013). In contrast, in another study IKdr as well as INa were inhibited by VIP (Pakhotin et al. 2006).
Interestingly, not only transient, acute effects were reported for VIP, but also long-lasting effects of
several hours duration (Kudo et al. 2013). Thus, there is an astounding similarity between VIP and
PDF, not only because both peptides are necessarily required for synchronization, but also because
apparently similar signal transduction cascades are employed and several ion channels might be
targeted.
PDF-dependent activations as well as inhibitions of the electrical activity were previously detected in
extracellular recordings of the isolated AME, but only inhibitions were observed in the absence of
synaptic transmission. Thus, the direct effect of PDF was suggested to be inhibitory (with respect to
cell excitability; i.e. hyperpolarizing), and the observed activations were suggested to be
disinhibitions (Schneider and Stengl 2005). However, since network activity of several AME neurons
was analyzed, it was complicated to draw clear conclusions on the single cells. Therefore, single AME
neurons in primary cell cultures were analyzed. In the patch clamp experiments different whole-cell
current components (IK, INa, ICa, Iin,sust) were mainly inhibited by PDF. Other currents such as the
presumptive non-selective cation current were activated. Most probably IK and Iin,sust themselves
consisted of different current components, such as IKdr, IK(Ca), Ca2+ currents, and probably IK(Na) and ICl.
191
Discussion
Thus, further pharmacological experiments might be performed, to reveal the exact ion channels,
which are PDF-dependently modulated. So far, it seems that both inhibition (hyperpolarization) and
activation (depolarization) of the cell excitability are direct effects of PDF, which was also indicated in
Ca2+ imaging experiments on single AME neurons (Wei et al. 2014). Depolarizations could be caused
by inhibition of different K+ currents or activation of non-specific cation currents, resulting in an
inward current at hyperpolarized membrane potentials, while hyperpolarizations could be caused by
inhibition of Na+ currents or Ca2+ currents (Fig. 117). A contribution of Cl- currents is unlikely, since
PDF effects in extracellular recordings were still observed in the presence of PTX (Schneider and
Stengl 2005). In the extracellular recordings performed for this thesis, PDF-dependent oscillations of
the electrical activity or bursting of AME neurons were observed for the first time (see 3.1.3, 4.1.3).
This could be mediated by parallel depolarizing and hyperpolarizing effects in the same cell, such as
block of K+ currents and Na+ currents (Fig. 117). Thus, PDF-dependent ultradian membrane potential
oscillations might be a prerequisite for synchronization of PDF-sensitive cells (Wei et al. 2014). If the
PDF-sensitive cells belong to different assemblies, they may form a new PDF-dependent assembly, as
suggested by Schneider and Stengl (2005). When two PDF-sensitive cells with synaptic connection
are synchronized, synaptic transmission between these cells might be improved via resonance
(Izhikevich et al. 2003; Schneider and Stengl 2005). If the PDF-releasing cell has PDF autoreceptors, as
shown for different clock neurons of D. melanogaster (Shafer et al. 2008; Lear et al. 2009; Im and
Taghert 2010; Kula-Eversole et al. 2010; Im et al. 2011; Choi et al. 2012), all three cells could resonate
in synchrony (Fig. 117).
In D. melanogaster the PDF-dependent reset of the molecular feedback loop involves stabilization of
both PER and TIM in a cAMP- and PKA-dependent manner (Li et al. 2014a; Seluzicki et al. 2014).
Similar results were obtained in a modeling study, based on the D. melanogaster feedback loop
components, which were known at that time (Petri and Stengl 2001). It was suggested that PDF
inhibits phosphorylation events and activates dephosphorylation events of PER and TIM, which
should also result in a stabilization of these core clock loop components. So far, no experimental
studies were performed to analyze PDF effects on clock proteins in R. maderae. However, PDF was
shown to phase-shift the circadian locomotor activity resulting in a PCR with prominent phase-delay
in the late subjective day/early subjective night and mild phase-advance in the late subjective
night/early subjective day (Schendzielorz 2013). It seems likely, that activation (depolarization) might
result in a phase advance and inhibition (hyperpolarization) in a phase delay. Given that PDF signals
most probably via Gαs in most AME neurons, the cAMP-dependent PKA could be involved in both
kinds of phase shifts. Since depolarizations but not hyperpolarizations should be accompanied by
increases of [Ca2+]i probably the Ca2+-dependent PKC might be activated next to PKA. Thus,
phosphorylation events by both PKA and PKC could possibly mediate the phase advance (Wei et al.
2014). However, it is beyond the scope of this thesis to speculate about the transduction of PDFeffects to the molecular feedback loop, since the experiments focused on electrogenic effects of PDF.
192
Discussion
Fig. 117. Hypothetical model for PDF-signaling in spontaneously active AME cells. A. The PDF receptor (PDFR) is
suggested to couple to an adenylyl cyclase (AC)-stimulating G protein (Gs) in most AME neurons. PDF binding is suggested
to activate the Gs protein, leading to a separation of the βγ-subunit from the Gαs-subunit, which activates adenylyl cyclase.
PDF-dependent increases of the cAMP level possibly activate protein kinase A (PKA). cAMP-dependent activation of HCN
+
2+
channels and cAMP- or PKA-dependent block of K channels depolarize the neuron, leading to a subsequent rise of [Ca ]i.
2+
If protein kinase C (PKC) is Ca -dependently activated, both PKA and PKC might affect the transcriptional translational
2+
feedback loop (TTFL) and phase-advance the cell. B. PDF-dependent hyperpolarizations could be mediated via block of Ca
+
2+
and Na channels, either cAMP- (not shown) or PKA-dependently. Since [Ca ]i decreases PKC is not activated, and PKA
alone might phase-delay the TTFL. C. Simultaneous occurrence of hyperpolarizing and depolarizing effects of PDF in one
neuron is suggested to mediate rhythmic membrane potential oscillations and switch the neuron to bursting mode. PDF
might synchronize several PDF-sensitive, spontaneously active cells in vicinity to the PDF release sites. If the PDF releasing
+
cell (PDF ) expresses PDF autoreceptors, it is also affected by its own PDF release. In the model, PDF phase-locks
membrane potential oscillations of all three cells. Hypothetical synaptic transmission between the PDF releasing cell,
cell A, and cell B are improved, and the cells resonate in synchrony. Figure redrawn and modified after (Wei et al. 2014).
193
Discussion
4.3 Analysis of peripheral pacemaker neurons in M. sexta
antennae
The characterization of central pacemaker neurons was performed with AME neurons of the
cockroach R. maderae. Among the variety of peripheral pacemakers, olfactory receptor neurons
(ORNs) were chosen, which control the daytime-dependent sensitivity of the insects' antennae. Since
olfaction is best characterized for D. melanogaster and different moths, and primary ORN cell
cultures of the hawkmoth M. sexta have been established, M. sexta seemed to be better suited than
R. maderae for the investigation of peripheral pacemaker neurons.
A good proportion of research in insect olfaction of the last decade was focused on the olfactory
coreceptor ORCO. ORCO is essential for OR-dependent olfaction in D. melanogaster, since it is
required for trafficking and maintenance of conventional ORs to/in the plasma membrane of the
ORNs' outer dendrites, where olfactory signal transduction takes place (Benton et al. 2006).
Consistently, ORCO null mutant flies and flies, in which ORCO expression was knocked down via
RNAi, showed severe olfactory impairments (Larsson et al. 2004; Neuhaus et al. 2005; Benton et al.
2006). As a prerequisite for trafficking ORCO heteromerizes with conventional ORs (Neuhaus et al.
2005; Benton et al. 2006; German et al. 2013). In heterologous expression systems, such as
mammalian cell lines or X. laevis oocytes, these heteromers function as odorant gated ion channels,
whereby the stoichiometry and the location of the pore is not known yet (Sato et al. 2008; Wicher et
al. 2008). Next to the ionotropic activation mechanism, ORCO is also activated by cyclic nucleotides
and phosphorylation by PKC (Wicher et al. 2008; Sargsyan et al. 2011). The experiments in chapter
3.3 focused on a characterization of the M. sexta ORCO orthologue. It was hypothesized that
MsexORCO functions as pacemaker ion channel in vivo, which regulates membrane potential and
calcium concentration of the ORNs instead of mediating the olfactory transduction current.
4.3.1 Transiently transfected HEK 293 cells poorly expressed ORCO
The first characterization of MsexORCO was performed in a heterologous expression system. Initially,
HEK 293 cells were transiently transfected with MsexOrco and used for immunocytochemistry. Only
about 7.0 % of the cells were ORCO-ir. Since neither non-transfecting control cells nor omitting the
primary or secondary antibody revealed any ORCO-ir cells, the staining appeared to be specific. The
transfection efficiency did not depend on the transfection reagent or a possible cotransfection of
MsexSnmp-1 and/or one of the pheromone receptor candidates MsexOr-1 or MsexOr-4. In another
study HEK 293T cells were transiently transfected with DmelOrco and DmelOr-22a, which were both
gfp-tagged, with a transfection efficiency of 52 % (Ignatious Raja et al. 2014). Even if this value
reports the combined expression of ORCO and OR-22a, the transfection efficiency was far better than
the transfection efficiency detected here. The reasons for this discrepancy could lie in differences in
the HEK cell line, the transfection reagent, or simply in the different proteins. It seems likely, that
different ORCO orthologues or ORs are more or less suitable for expression in a certain foreign cell
type.
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Discussion
ORCO expression was not affected by cotransfection with or-1/4 or snmp-1. In contrast, in
D. melanogaster ab3A neurons the abundance of ORCO was shown to be reduced in the absence of
OR-22a/b, indicating a stabilizing function of the coexpressed ORs (Benton et al. 2006). However, it is
not convenient to compare such in vivo properties with the situation in the heterologous system, and
other studies using heterologous expression systems did not report about possible effects of OR
coexpression on ORCO expression. Due to the "chaperone-function" of ORCO other studies rather
focused on the effects of ORCO coexpression on the expression level of ORs. In my thesis the
transfection efficiency of or-1/4 and snmp-1 could not be determined, since no specific antibodies
were available. Attempts to detect the pheromone receptor candidates with an antibody directed
against the bombykal receptor of B. mori (BmorOR-3), with an epitope differing in six of 16 amino
acids for MsexOR-1 and twelve of 16 amino acids for MsexOR-4, failed.
Another problem next to the low expression efficiency was the membrane insertion of ORCO. The
majority of ORCO-ir cells appeared to express ORCO rather in the cytosol than in the plasma
membrane. DAPI staining revealed huge nuclei, which took up the major part of the HEK 293 cells,
leaving only little place for cytosol, which was completely ORCO-ir, whereas it cannot be excluded
that these proportions were artifacts of the staining procedure. Although cells were detected, in
which the ORCO-immunoreactivity largely overlapped with the WGA signal used for membrane
labeling, this cannot prove, but at best suggest membrane insertion. Depending on numerical
aperture of objective and condenser and the wavelength of the light, the resolution of light
microscopes is limited to values around 200 nm (Abbe-limit). Even if the resolution might be slightly
increased for confocal fluorescence microscopy, it is not possible to resolve a cell membrane with a
thickness of 5 - 10 nm (Alberts et al. 2007). In future experiments membrane insertion should be
detected with biochemical methods, e.g. by labeling the cell surfaces, separating the labeled
membranes from the residual components of the cells, and performing western blots with the
fractions (Cole et al. 1987). The availability of an specific ORCO antibody, detecting one of the
extracellular loops, could also enormously facilitate the detection: in the absence of detergents such
an antibody should exclusively detect membrane-inserted ORCO from the extracellular side.
However, localization of ORs and ORCO orthologues from other insects within the cytoplasm was
also observed in other studies using HEK 293T cells (Ignatious Raja et al. 2014) and even insect cell
lines (German et al. 2013). Since a large amount of the proteins was still detected in isolated
membrane fractions in these studies, it was suggested that the proteins reside mainly in intracellular
membranes (German et al. 2013). It seems likely that some factors, which are crucial for membrane
trafficking of ORCO or ORs, are missing in different heterologous systems. It would be interesting for
example, to examine the effects of coexpressed G protein coupled receptor kinase 2 (GPRK2), which
was shown to control rhythmic expression of OR-22a in the dendrites of ab3A neurons of
D. melanogaster (Tanoue et al. 2008).
During writing of this thesis the published sequence of the MsexOrco gene (Grosse-Wilde et al. 2010)
was found to be incomplete (personal communication with Dieter Wicher, Max Planck Institute for
Chemical Ecology, Jena). Therefore, all experiments were performed with a shortened version of the
MsexORCO protein, lacking the first 37 amino acids of the N-terminus. Recently, functionally
important amino acids of DmelORCO were identified in a cluster in the N-terminal tail (within the
first 37 amino acids), in the extracellular loop 2, and in the intracellular loop 3 (Hopf et al. 2015). For
a closer investigation of the N-terminal cluster of amino acids, mutant proteins bearing mutations of
six amino acids or a deletion of eleven amino acids of this cluster were generated and expressed in
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Discussion
X. laevis oocytes. Remarkably, the cells did not respond to the ORCO agonist VUAA1 and showed
highly reduced or abolished odorant responses when the mutant DmelORCO proteins were
coexpressed with a tuning OR. It was hypothesized that the N-terminus might be functionally
important for folding, trafficking, or complex formation (Hopf et al. 2015). Thus, it seems likely that
the low expression rate of MsexORCO was due to the missing amino acids in the N-terminal tail of
the protein.
4.3.2 Transiently transfected HEK 293 cells did not reliably respond
to pheromone stimulation
To investigate, whether MsexORCO mediates responses to (E,Z)-10,12-hexadecadienal (bombykal,
the main component of M. sexta's pheromone blend) in heterologous expression systems, one of
two male-specific ORs was coexpressed with MsexORCO: MsexOR-1 or MsexOR-4. The closest
relative of MsexOR-1 is BmorOR-3, the receptor detecting bombykal in B. mori, and the closest
relative of MsexOR-4 is HvirOR-13 (HR13), the receptor detecting Z11-hexadecenal, the main
pheromone component of H. virescens (Grosse-Wilde et al. 2010). The ligands of both pheromone
receptor candidates were unknown.
When HEK 293 cells, transfected with MsexOrco and MsexOr-1/4, were stimulated in calcium imaging
experiments with bombykal, in most experiments no reproducible, stimulus-correlated [Ca2+]
increases were detected. The few observed [Ca2+] increases differed in their kinetics and had delays
and durations in the range of tens of seconds to minutes. Even when the kinetics were not taken into
consideration and all [Ca2+] increases after stimulation were counted, stimulation with bombykal at
concentrations of 10-15 - 10-9 M was not significantly different from control experiments. Only
stimulation of MsexOrco/MsexOr-1-cotransfected cells with the highest concentration of bombykal
(10-6 M) evoked a significantly higher percentage of active cells compared with lower concentrations
or solvent control applications. Considering the high sensitivity of pheromone detection in vivo,
micromolar concentrations of bombykal seem to exceed the physiological range and might produce
unspecific responses. The lack of responsiveness to lower bombykal concentrations could be due to
several reasons: (i) MsexORCO and/or MsexOR-1/4 could be in a desensitized state; (ii) in contrast to
ORCO orthologues of other insect species MsexORCO might not act as ion channel and thus not
mediate pheromone-gated responses, when expressed together with pheromone receptors in
heterologous systems; (iii) other crucial parts of the signaling cascade could be missing; (iv) DMSO,
which was used as solvent, might not substitute the pheromone binding protein, which is required
for pheromone responses in vivo; (v) both pheromone receptor candidates might detect different
pheromone components than bombykal; (vi) the percentage of cells showing membrane insertion of
MsexORCO and MsexOR-1/4 might be too low. Therefore different strategies were followed to
analyze the low bombykal sensitivity.
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Discussion
4.3.2.1 CaM-dependent desensitization appears unlikely
A possible desensitization of proteins mediating Ca2+ influx could be mediated by calmodulin (CaM).
CaM is one of the most abundant Ca2+ sensors in eukaryotic cells. It is activated by Ca2+ binding and
mediates different processes to regulate the Ca2+ homeostasis: CaM activates Ca2+/CaM-dependent
protein kinases and the Ca2+-ATPase, which pumps Ca2+ out of the cell. Furthermore, it modulates
target ion channels (Alberts et al. 2007). In vertebrate ORNs for example, CaM is implicated in the
fast desensitization of CNG channels and the extrusion of Ca2+ by Ca2+-ATPases (Song et al. 2008;
Antolin et al. 2010). Consistently, MsexORCO and different other orthologues were shown to possess
a conserved CaM binding motif (Mukunda et al. 2014). In the present experiments the CaM inhibitor
W7 was used to examine a possible CaM modulation of ORCO. Since neither spontaneous [Ca2+]
increases after W7 treatment nor bombykal-dependent [Ca2+] increases were detected, a CaMdependent desensitization of MsexORCO appears unlikely. Surprisingly, DmelORCO-mediated [Ca2+]
increases were previously shown to be decreased and prolonged by CaM inhibition or mutation of
the CaM binding motif, suggesting a role of CaM in sensitization but not desensitization (Mukunda et
al. 2014; Mukunda Shivalingaiah 2014; Review: Wicher 2015). Considering that the effects of CaM on
DmelORCO were dependent on the identity of the coexpressed OR (Mukunda et al. 2014), and that
the experiments performed for this thesis were limited to MsexOR-1 coexpression, no further
conclusions might be drawn. However, the lack of responses after CaM inhibition are rather in
accordance with a role of CaM in sensitization than in desensitization.
4.3.2.2 Replacement of MsexORCO by other ORCO orthologues or Gα15 did not
improve the response rate to bombykal
To analyze whether the lack of bombykal responses was due to a non-ion channel function of
MsexORCO, it was tested whether a replacement of MsexORCO in the heterologous expression
system might improve the bombykal responsiveness. Different studies performed on
D. melanogaster (Jones et al. 2005) or heterologous expression systems (Nakagawa et al. 2005;
Nichols et al. 2011) reported that ORCO orthologues of different insect species may functionally
substitute for each other. Therefore, MsexOR-1 was coexpressed with ORCO orthologues, for which
the ion channel function in heterologous systems was clearly demonstrated before. Replacement of
MsexORCO by DmelORCO seemingly did not improve the bombykal responsiveness. In a second
approach, the putative pheromone receptors were expressed in SF9 cells. This insect cell line was
shown to express endogenous ORCO and to mediate odorant-gated responses, when DmelOR-43b or
DmelOR-22a were expressed without coexpression of DmelORCO (Kiely et al. 2007; Smart et al.
2008). Additionally, SF9 cells should offer a cellular environment that is less artificial than that of the
mammalian HEK 293 cells. However, in this experimental series no significant bombykal responses
were detected, neither for MsexOr-1 nor for MsexOr-4 transfected cells. In the third approach, an
ORCO independent signaling system was used, the murine G protein α-subunit Gα15, which was
shown to couple a variety of receptors, amongst others insect ORs, to PLC signaling (Offermanns and
Simon 1995; Wetzel et al. 2001). Activation of the receptor theoretically should results in an increase
of IP3 and a subsequent [Ca2+] increase via activation of IP3 receptors of the endoplasmatic reticulum
(Wetzel et al. 2001). Although this system was reported several times to mediate reliable odorant
197
Discussion
responses in heterologous systems (Grosse-Wilde et al. 2006; Grosse-Wilde et al. 2007; Pregitzer et
al. 2014), only a few cells cotransfected with gα15 and MsexOr-1 or MsexOr-4 showed [Ca2+] increases
after bombykal stimulation and the percentages of active cells did not differ from control
applications. Thus, all approaches, in which MsexORCO was replaced either by other ORCO
orthologues or different signaling components, which were known to mediate odorant responses in
heterologous systems, failed to improve the bombykal responsiveness. Even though the number of
experiments was very small for all three approaches, these experiments suggested, that the low
bombykal sensitivity was not due to a non-ion channel function of MsexORCO, but rather to other
reasons. The missing amino acids in the N-terminal tail of MsexORCO most probably did not impair
its ion channel function since the majority of amino acids, whose mutation affected the ion channel
function of other ORCO orthologues, were located in a region between the fifth and the seventh
transmembrane domain, but not in the N-terminal tail (Hopf et al. 2015).
4.3.2.3 Coexpression of SNMP-1 did not specifically improve the bombykal
responsiveness
To test whether the low response rate to bombykal was due to the absence of a crucial factor of the
pheromone transduction cascade, the sensory neuron membrane protein 1 (SNMP-1) was
coexpressed with ORCO and OR-1/4. In different moth species as well as D. melanogaster, two
subtypes of SNMP (SNMP-1 and SNMP-2) are expressed in the antennae and other sensory organs
(Rogers et al. 1997; Rogers et al. 2001a; Rogers et al. 2001b; Benton et al. 2007). In D. melanogaster
the SNMP-1 subtype (usually just termed DmelSNMP) is coexpressed with OR-67d, the receptor
detecting the aggregation pheromone 11-cis-vaccenyl acetate (cVA, Ha and Smith 2006; Kurtovic et
al. 2007), in ORNs of trichoid sensilla (at1). Additionally, it is expressed in supporting cells throughout
the antenna (Benton et al. 2007). In contrast, in the moths H. virescens and A. polyphemus the
SNMP-1 subtype is expressed in pheromone-sensitive ORNs of trichoid sensilla and the SNMP-2
subtype is expressed in the supporting cells of these sensilla (Forstner et al. 2008). In M. sexta nearly
identical expression patterns of SNMP-1 and SNMP-2 were found. Both subtypes were reported to be
localized in trichoid as well as basiconic sensilla, most probably in ORNs, and the expression occurred
simultaneously with the onset of olfactory function (Rogers et al. 2001a). However, studies with
higher resolution are required to confirm the spatial expression pattern. SNMPs belong to the CD36
family, whose members are characterized by two transmembrane domains and function in binding
and transport of lipids and proteinaceous ligands, suggesting that SNMPs are potentially involved in
the recognition of the lipophilic pheromone components (Bonen et al. 2004; Review: Vogt et al.
2009). When DmelSNMP-1 or MsexSNMP-1 were coexpressed with MsexORCO and MsexOR-1 or
OR-4, no improvement of the bombykal response rate was observed. Although more cell transfected
with orco, or-1, and DmelSnmp-1 than cells transfected with orco, or-4, and DmelSnmp-1 responded
to bombykal, no significant differences were detected between bombykal stimulations and the
respective control experiments, neither for OR-1 nor for OR-4 expressing cells. Thus, SNMP-1 did not
appear to be the missing crucial factor. Remarkably, the percentage of active cells was slightly
elevated by SNMP-1 coexpression, not specifically for bombykal stimulations but also for solvent
control applications or spontaneous [Ca2+] increases. This suggests, that the incorporation of SNMP-1
in the membrane elevated the sensitivity as well as the spontaneous [Ca2+] increases in an unspecific
and unknown manner. In contrast, in D. melanogaster the presence of SNMP-1 was shown to be
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Discussion
required for the detection of low concentrations of cVA by OR-67d neurons or the detection of Z11hexadecenal by OR-67d neurons misexpressing the HR13 receptor (Benton et al. 2007; Jin et al.
2008). Furthermore, it was demonstrated to be necessary for rapid activation and termination
kinetics of the cVA responses in vivo (Li et al. 2014b), while an absolute requirement for cVA
detection and an SNMP-dependent inhibition of spontaneous activity in OR-67d neurons are
currently under debate (Benton et al. 2007; Jin et al. 2008; Li et al. 2014b). In other heterologous
expression studies the coexpression of H. virescens SNMP-1 increased Z11-hexadecenal sensitivity of
HR13 expressing cells by the factor of 1000 (Pregitzer et al. 2014). The detailed mechanism of the
SNMP-1 function is still unknown, but it was hypothesized that it decreases an energy barrier
between the hydrophilic sensillum lymph and a lipophilic pheromone binding site, which has to be
surmounted during association and dissociation of pheromone and receptor (Li et al. 2014b). A direct
interaction between SNMP-1 and the receptor complex was not demonstrated so far. When possible
interactions between heterologously expressed SNMP subtypes, ORCO, and OR-22a were
investigated in FRET experiments, no interaction between ORCO and SNMP-1 or SNMP-2, but
between OR-22a and SNMP-1 was detected (German et al. 2013). However, since OR-22a does not
seem to be coexpressed with SNMP-1 in vivo, the significance of this interaction is questionable, but
the lack of interaction with ORCO suggests that the possible interaction site is rather the pheromone
receptor than ORCO. This point of view is also supported by experiments, in which SNMP increased
the sensitivity of Z11-hexadecenal responses of HR13, which was coexpressed with Gα15 instead of
ORCO (Pregitzer et al. 2014). The discrepancy between the pheromone response promoting effects
of SNMP-1 in other studies and the inefficacy of SNMP-1 coexpression in the present experiments
indicates that SNMP-1 expression might increase the sensitivity of functional pheromone receptor
complexes in heterologous expression systems, but is not sufficient to ensure the functionality of
otherwise non-functional receptor complexes.
Recently, another factor was described, which is required for robust odorant responses in ORCO/OR
expressing neurons of D. melanogaster, the P4-type ATPase (a phospholipid flippase) dATP8b. The
flippase is required to maintain an asymmetric phospholipid composition between exoplasmatic and
cytoplasmatic leaflets of the plasma membrane (Liu et al. 2014b), which appears to be a crucial
requirement for membrane localization of specific ORs, such as the pheromone receptor OR-67d, but
not for ORCO (Ha et al. 2014). Thus, in future experiments one could test the effects of coexpression
of an P4-type ATPase. However, since dATP8b function could be rescued by expression of a bovine
P4-type ATPase (Ha et al. 2014), and different P4-type ATPases are expressed in human kidney tissue
(Folmer et al. 2009; Coleman et al. 2013), it seems likely that HEK 293 cells express an endogenous
P4-type phospholipid flippase, which is sufficient.
4.3.2.4 BSA as solvent did not improve the response rate to bombykal but
caused OR/ORCO-independent [Ca2+] increases
The next factor of uncertainty that was examined was the compound, which was used to dissolve the
lipophilic pheromone component. When pheromones are detected in vivo, the molecules enter the
aqueous sensillum lymph via pores in the cuticula and are subsequently bound by pheromone
binding proteins (PBPs) or odorant binding proteins (OBPs, Vogt and Riddiford 1981; Review: Venthur
et al. 2014). The PBPs are abundant proteins in the sensillum lymph, which are present at
199
Discussion
concentrations up to 10 mM (Klein 1987), and are required for solubilization and transport of
pheromone components and probably also for specific interactions with the pheromone receptors
(van den Berg and Ziegelberger 1991; Forstner et al. 2006; Grosse-Wilde et al. 2006; Forstner et al.
2009). Additionally, a function in scavenging and inactivation of the pheromones has been proposed
(Vogt and Riddiford 1981; Kaissling 1986). In the majority of experiments DMSO was used to dissolve
bombykal since different studies suggested its sufficiency for solubilization and mediation of
pheromone responses in heterologous systems (Nakagawa et al. 2005; Grosse-Wilde et al. 2006).
Even the continuous presence of DMSO in the bath did not improve the cells' sensitivity to bombykal.
Although DMSO is commonly used as solvent, it was also reported that the sensitivity of pheromone
detection in heterologous systems was increased by the use of PBP instead of DMSO (Forstner et al.
2009). Since no M. sexta PBP was available, the possible effects of DMSO substitution by BSA were
tested. BSA binds fatty acids and other lipophilic components (Kragh-Hansen 1981; Peters Jr 1985;
van den Berg and Ziegelberger 1991). It was shown to completely substitute the effects of PBPs in
different studies (van den Berg and Ziegelberger 1991; Stengl et al. 1992b; Kaissling 2013). However,
HEK 293 cells responded with strong OR/ORCO-independent [Ca2+] increases to BSA stimulation.
These responses were mainly due to BSA-bound fatty acids, but BSA itself or other BSA-bound
components also caused [Ca2+] increases. Different BSA-dependent Ca2+ influx pathways were
demonstrated for various cell types before, such as increased Ca2+ currents in chicken granulosa cells
(Chiang et al. 1993), increased LVA Ca2+ currents in NG108-15 cells (Schmitt and Meves 1994), or
CATSPER-dependent Ca2+ influx in mouse sperm (Xia and Ren 2009). Likewise, ion channel
modulation by (long chain) fatty acids, which might be bound by BSA, has been well known for a long
time (Reviews: Ordway et al. 1991; Meves 2008). In the specific case of HEK 293 cells, arachidonic
acid-activated, non-store operated Ca2+ entry (Holmes et al. 2007) or lysophosphatidic acid-activated,
IP3-dependent calcium increase (van Corven et al. 1989; Alderton et al. 2001; Eiras et al. 2004) were
reported. Since HEK 293 cells also express different TRP channels (Garcia and Schilling 1997;
Groschner et al. 1998; Bugaj et al. 2005; Zagranichnaya et al. 2005), another conceivable mechanism
for the observed [Ca2+] increases might be the activation of TRP channels by polyunsaturated fatty
acids (PUFAs), as suggested for D. melanogaster TRP channels (Chyb et al. 1999; Leung et al. 2008).
However, since BSA-dependent [Ca2+] increases were not in the focus of the present experiments,
the mechanism was not further characterized. The lack of significant bombykal effects, when fatty
acid-free BSA was used as solvent, contradicts the idea, that the low bombykal sensitivity was due to
ineffective solubilization of the pheromone component by DMSO, but it has to be noted that a
possible improvement of the sensitivity by the presence of a specific PBP could not be excluded and
should be tested in future experiments.
4.3.2.5 The response rate to C-15 was very low
To test, whether MsexOR-1 or MsexOR-4 detect other pheromone components than bombykal,
HEK 293 cells cotransfected with orco, or-1/4, and snmp-1 were stimulated with low concentrations
(10-14 - 10-12 M) of (E,Z)-11,13-pentadecadienal (usually referred to as C-15). C-15 simulates (E,E,Z)10,12,14-hexadecatrienal, another component of the pheromone blend (Christensen and Hildebrand
1987; Christensen et al. 1989; Kaissling et al. 1989; Christensen and Hildebrand 1997). This
component was chosen, because (E,E,Z)-10,12,14-hexadecatrienal and bombykal were shown to be
required for full behavioral pheromone responses, while all other components played only minor
200
Discussion
roles (Tumlinson et al. 1989). As shown for bombykal stimulation, the response rate was very low
and usually not significantly different from control applications or spontaneous [Ca2+] increases. Only
in a subset of the experiments, which were performed with orco/or-4/snmp-1-cotransfected cells in
the continuous presence of DMSO in the bath, the percentage of active cells after C-15 stimulation
was higher than the percentage of cells showing spontaneous [Ca2+] increases, suggesting that
MsexOR-4 might be the receptor detecting (E,E,Z)-10,12,14-hexadecatrienal in vivo. Since the closest
relative of MsexOR-4 is the H. virescens receptor (HR13) detecting Z11-hexadecenal (Grosse-Wilde et
al. 2007; Grosse-Wilde et al. 2010), which is one of the minor components of M. sexta's pheromone
blend, this component should be tested in future experiments.
4.3.2.6 Apparently, the low response rate to pheromone stimulation was due
to the low expression of MsexORCO, MsexOR-1, and MsexOR-4
Different strategies were followed to characterize the low response rate of HEK 293 cells
cotransfected with orco and or-1/4 to pheromone stimulation, namely CaM inhibition, substitution of
MsexORCO (by DmelORCO or Gα15 or by expression in SF9 cells), coexpression of SNMP-1,
replacement of the solvent, and stimulation with a different pheromone component. All these
modifications did not increase the low response rate, indicating that the reason most probably was
the low expression of the pheromone receptor candidates. In contrast to ORCO expression,
expression of OR-1 and OR-4 could not be analyzed since no specific antibody was available. Thus, it
must be assumed that the transfection rate for the presumptive pheromone receptors was not
higher than the transfection rate for orco (about 7.0 %). Since ORCO-immunoreactivity was detected
usually in the cytosol and membrane insertion was not reliably confirmed, one must assume that a
large proportion of the ORCO-expressing cells did not functionally express ORCO in the membrane
(see 4.3.3). In contrast to the situation in vivo, ORCO is not absolutely required for membrane
insertion of ORs in heterologous expression systems (Wetzel et al. 2001; Nakagawa et al. 2005;
Grosse-Wilde et al. 2006; Wicher et al. 2008). However, even if the percentage of expression and
membrane insertion was higher for OR-1/4 than for ORCO, it must be assumed that OR expression in
the absence of ORCO or other coexpressed signaling molecules such as Gα15 usually was not
functional. Therefore, the population of cells functionally expressing both ORCO as well as a
pheromone receptor candidate is most probably only a subpopulation of the cells showing functional
ORCO expression, which perfectly explains the low response rate to pheromone stimulation. In rare
cases the expression of an odorant receptor alone might be sufficient for odorant responses in
heterologous expression systems, as reported for HEK 293 cells expressing DmelOR-22a or DmelOR43a, suggesting that insect ORs might couple by chance to endogenous G proteins of HEK 293 cells.
However, millimolar odorant concentrations were required for response rates below 1 % (Neuhaus
et al. 2005). In the present experiments the maximum bombykal concentration was 10-6 M, which at
the same time was the only effective concentration for OR-1-mediated responses. Since pheromone
transduction is a highly sensitive process in vivo and moths were suggested to be able to detect
single pheromone molecules (Kaissling and Priesner 1970), unspecific effects should be avoided and
in most experiments pheromone concentrations between 10-15 and 10-9 M were employed. Except
for one study, in which HR13-SNMP-1- expressing cells significantly responded to Z11-hexadecenal at
a concentration of 10-15 M (Pregitzer et al. 2014), most studies employing heterologous expression
systems demonstrated the requirement of micromolar or even millimolar odorant concentrations
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Discussion
(Nakagawa et al. 2005; Neuhaus et al. 2005; Sato et al. 2008; Smart et al. 2008; Jones et al. 2011;
Nichols et al. 2011; Pask et al. 2011; Chen and Luetje 2012; Nakagawa et al. 2012). Although the
receptor complexes should be overexpressed in these studies, the artificial environment and the lack
of crucial signaling molecules seems to dramatically decrease the odorant sensitivity in comparison
with in vivo conditions. Therefore, it should be considered for future experiments to stimulate the
cells with pheromone concentrations higher than 10-6 M, to obtain more reliable results. The present
results hint that MsexOR-1 could be the receptor detecting bombykal and MsexOR-4 the receptor
detecting (E,E,Z)-10,12,14-hexadecatrienal in vivo, but due to the low expression and thus the low
response rate it was not possible to unequivocally identify the specific ligands for both receptors.
Likewise, the low response rate did not allow to answer the question whether MsexORCO mediates
pheromone responses when coexpressed with a pheromone sensing receptor in heterologous
expression systems.
4.3.3 The majority of transiently transfected HEK 293 cells did not
respond to ORCO modulation
4.3.3.1 MsexORCO appears to function as Ca2+ permeable ion channel, which
is activated by VUAA1
The hypothetical receptor complex of MsexORCO and MsexOR-1/4 might be activated either by
pheromone binding to OR-1/4 or by direct modulation of ORCO. Since it was not possible to
characterize the function of MsexORCO indirectly via pheromone stimulation (see 4.3.2), ORCO was
activated directly. Therefore, the ORCO agonist VUAA1 was employed (Jones et al. 2011). In these
experiments VUAA1 stimulation evoked specific, ORCO dependent [Ca2+] increases in 3.0 % of the
cells (median), suggesting that this percentage equals the percentage of cells with functional
MsexORCO expression. Thus, less than 50 % of the cells showing ORCO-immunoreactivity (median:
6.4 %) appeared to express MsexORCO at the cell surface, supporting the former speculations about
the low pheromone response rate of the cells (see 4.3.2). Apparently, the missing amino acids in the
N-terminal tail of MsexORCO affected the expression as well as the membrane localization of the
protein. It cannot be excluded that its ion channel function was also impaired. However, as
mentioned above, the C-terminal region but not the N-terminal region of other ORCO orthologues
was suggested to be important for the ion channel pore (Hopf et al. 2015). The demonstration of
VUAA1-dependent [Ca2+] increases extended the agonistic spectrum of VUAA1 to another ORCO
orthologue and suggested that MsexORCO indeed functions as ion channel, which mediates Ca2+
influx. However, direct evidence for an ion channel function of MsexORCO could not be provided. For
this purpose further experiments are required. Ca2+-free ringer solution could be used to confirm that
VUAA1 mediated [Ca2+] increases indeed depend on Ca2+ influx from the extracellular solution.
Additionally, patch clamp recordings should be performed to confirm MsexORCO-dependent single
channel or whole-cell currents and to analyze the ion selectivity of the channel (Wicher et al. 2008).
Even though the VUAA1 dependent [Ca2+] increases were significantly different from control
experiments, the response rate (median: 3.0 %) was still too low to facilitate a detailed investigation
of the mechanism.
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Discussion
4.3.3.2 Effects of coexpressed ORs on the VUAA1 sensitivity
Jones et al. reported that the VUAA1 sensitivity is higher for heteromeric receptor complexes
(AgamORCO + AgamOR-10) compared to homomeric ORCO complexes (2011). In the present
analysis, it was not discriminated between transfection of orco alone or cotransfection of orco,
or-1/4, and snmp-1. Therefore, a possible influence of the pheromone receptor candidates and
SNMP-1 to the VUAA1-dependent [Ca2+] increases was not characterized. However, in experiments
performed by another researcher it was demonstrated that coexpression of MsexSNMP-1 and
MsexOR-1/4 significantly enhanced the VUAA1-dependent [Ca2+] increases, suggesting positive
interactions between the heterologously expressed proteins (Nolte et al. 2013). Probably SNMP-1
interacted with the pheromone receptors but not with ORCO, and the pheromone receptors
interacted with both ORCO and SNMP-1 (Neuhaus et al. 2005; Benton et al. 2006; German et al.
2013; Pregitzer et al. 2014). The detailed mechanism for the higher VUAA1 sensitivity remained
unclear. Since the presence of ORs has a weak stabilizing effect on ORCO (Benton et al. 2006), the
amount of functionally expressed ORCO might be higher. And since ORCO and ORs form homomeric
and heteromeric complexes with unknown stoichiometry (Neuhaus et al. 2005; Benton et al. 2006;
German et al. 2013), it seems likely that the presence of ORs promotes an ORCO-conformation with
increased VUAA1 accessibility or better Ca2+ conductance. Alternatively, the OR might contribute to
the pore of the ion channel and thus directly affect its properties (Sato et al. 2008; Nichols et al.
2011; Nakagawa et al. 2012).
4.3.3.3 MsexORCO mediated spontaneous [Ca2+] increases
In addition to the VUAA1 responses, MsexOrco transfected (orco-positive) cells showed more
spontaneous [Ca2+] increases than orco-negative cells, indicating that MsexORCO is spontaneously
active and thus might also mediate spontaneous activity in vivo (pacemaker function). As mentioned
before it was not discriminated between transfection of orco alone or cotransfection of orco,
or-1/4, and snmp-1. Therefore, the contribution of the pheromone receptor candidates and
SNMP-1 to the spontaneous [Ca2+] increases cannot be excluded. Spontaneous activity of OR/ORCO
complexes in heterologous expression systems was also demonstrated in other studies (Sato et al.
2008; Wicher et al. 2008; Pask et al. 2013). In one of these studies the spontaneous activity of
DmelOR-22a/DmelORCO complexes was reduced by an OR-22a inhibitor, suggesting that the OR unit
at least partially mediates the spontaneous activity of OR/ORCO heteromers (Wicher et al. 2008). In
another study employing heterologously expressed A. gambiae ORs the HMA-dependent reduction
of the baseline current was shown to depend on the tuning OR, indicating a function in spontaneous
activity modulation (Pask et al. 2013). In contrast, other studies demonstrated that cells transfected
with DmelOrco only had a higher resting current (Sargsyan et al. 2011) and a higher intracellular Ca2+
concentration than non-transfected control cells (Mukunda et al. 2014). Likewise, AgamORCO
expressing cells showed spontaneous single-channel currents (Jones et al. 2011), indicating
spontaneous ORCO activity independent of ORs. Several in vivo studies also dealt with the influence
of ORCO and ORs on spontaneous activity. ORNs of orco mutant flies and mosquitoes exhibited
strongly reduced spontaneous activity (Larsson et al. 2004; Benton et al. 2007; Deng et al. 2011;
203
Discussion
DeGennaro et al. 2013) and ORCO agonism/antagonism was shown to increase/decrease
spontaneous ORN firing in D. melanogaster (Su et al. 2012) and A. gambiae (Jones et al. 2011; Jones
et al. 2012), suggesting an ORCO-dependent regulation of spontaneous activity. However, since
membrane expression of the tuning ORs was also impaired in the absence of ORCO (Neuhaus et al.
2005; Benton et al. 2006), and deletion or replacement of tuning ORs changed the pattern of
spontaneous activity, apparently both ORCO and the tuning ORs contribute to spontaneous activity
in vivo. Therefore, it seems likely that coexpressed ORs and SNMP-1 (see 4.3.2) also contributed to
the spontaneous [Ca2+] increases.
4.3.3.4 MsexORCO appears to be activated by cAMP
Next to specific ORCO-agonism via substances of the VUAA1 family, DmelORCO was reported to be
sensitive to modulation via general second messenger pathways: DmelORCO was activated by cyclic
nucleotides (cAMP and cGMP) and by phosphorylation via PKC, the latter being a prerequisite for its
cAMP sensitivity. In heterologous expression systems basal PLC and PKC activity was sufficient for the
cAMP sensitivity, while high PLC or PKC activity led to an cAMP-independent activation of DmelORCO
(Wicher et al. 2008; Sargsyan et al. 2011). In the present experiments activation of PKC
(n = 5) or adenylyl cyclase (n = 8) did not lead to significantly more active MsexOrco-positive cells
than control applications. However, stimulation with the membrane permeable cAMP analogue
8-Br-cAMP evoked MsexORCO-specific [Ca2+] increases (n = 17). Similarly, the percentage of active
MsexOrco-positive cells after stimulation with 8-Br-cGMP (n = 25) was higher than after control
applications, but did not differ from MsexOrco-negative cells. When only paired data were taken into
account, the number of experiments was too low to detect significant responses for 8-Br-cAMP
(n = 9) or 8-Br-cGMP applications (n = 18). These results suggest a possible cyclic nucleotide
sensitivity of MsexORCO, similar to DmelORCO.
4.3.3.5 The amiloride derivatives HMA and MIA caused OR/ORCOindependent [Ca2+] increases in HEK 293 cells
Due to the low MsexORCO expression and thus the low response rate of possible agonists, it was not
feasible to perform reliable experiments, in which possible agonists were applied in the presence of
possible antagonists. Some experiments were performed to test whether possible antagonists might
block the basic activity of MsexORCO. Amiloride and different derivatives were reported to block
odorant gated currents of heterologously expressed OR/ORCO heteromers of different insect species
(Pask et al. 2013; Röllecke et al. 2013). In these experiments the amiloride derivatives HMA and MIA
turned out to be the most effective antagonists. Next to odorant gated currents, the baseline current
of AgamORCO + AgamOR-48 expressing cells as well as VUAA1 gated currents of cells expressing
different ORCO orthologues were blocked by HMA and MIA (Pask et al. 2013). In the calcium imaging
experiments both HMA and MIA were shown to evoke unspecific, MsexORCO-independent increases
of the F340/F380 fluorescence ratio, which prevented further experiments on fura-2-loaded HEK 293
cells employing these compounds (for further discussion on the effects of HMA and MIA see 4.3.5).
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Discussion
4.3.3.6 The insect repellent DEET does not appear to block MsexORCO
The next possible ORCO antagonist that was employed was the insect repellent DEET. It was shown
to inhibit odorant responses in D. melanogaster as well as A. gambiae at different levels: It reduced
odorant gated currents of different heterologously expressed OR/ORCO heteromers, inhibited
odorant gated neuronal responses in single sensillum recordings, as well as behavioral responses to
odorants. The requirement of ORCO for the widespread inhibitory effects suggested ORCO as
possible molecular target of DEET (Ditzen et al. 2008). In the present experiments cells transfected
with orco, or-1/4, and possibly snmp-1 were stimulated with DEET. No significant difference between
the percentages of active cells after DEET and control applications was detected. However, even
though not statistically significant, there was a tendency for a DEET-dependent activation, which
contradicts the possible antagonistic function. Indeed, different studies demonstrated controversial
results about the mechanism of DEET. One study identified a cholinesterase in insect and mammalian
neuronal systems as target of DEET (Corbel et al. 2009). Other studies could show that DEET is
detected at close range by the gustatory system and at long range by the olfactory system (Lee et al.
2010; DeGennaro et al. 2013). Next to DEET-dependent inhibition of responses to different odorants
(Davis and Sokolove 1976; Dogan et al. 1999; Ditzen et al. 2008; Bohbot and Dickens 2012; Röllecke
et al. 2013), DEET-dependent activation of ORNs in the absence of odorants was reported (Dogan et
al. 1999; Syed and Leal 2008; Xia et al. 2008; Liu et al. 2010), suggesting two possible mechanisms:
DEET could mediate its repellent effect either by blocking olfactory responses to attractive odorants
or by directly activating ORNs, which mediate avoidance. Apparently, DEET affects different
OR/ORCO complexes in distinct ways, leading to inhibition or activation depending on the identity of
the tuning OR and the odorant (Bohbot and Dickens 2010; Bohbot et al. 2011; Pellegrino et al. 2011).
Opposite effects depending on the tuning OR suggested the tuning OR instead of ORCO as target of
DEET. Indeed, a single amino-acid polymorphism in a tuning OR was sufficient to remove the
inhibitory effect of DEET (Pellegrino et al. 2011) and DEET was ineffective in blocking the ORCO
activating effect of ORCO-RAM-2, a substance of the VUAA1 family (Bohbot and Dickens 2012). In the
experiments of this thesis no inhibitory effect of DEET was detected, but there was a tendency for
activation.
4.3.3.7 No inhibitory effect of the cation channel blocker ruthenium red was
observed
Another possible ORCO antagonist tested was the cation channel blocker ruthenium red. Different
studies demonstrated the capability of ruthenium red to block odorant or VUAA1 gated currents as
well as [Ca2+] increases of cells heterologously expressing different OR/ORCO orthologues (Nakagawa
et al. 2005; Sato et al. 2008; Nichols et al. 2011; Pask et al. 2011; Mukunda et al. 2014). Additionally,
a reduction of the basal Ca2+ concentration in the absence of an agonist was shown (Sato et al. 2008).
Interestingly, the efficiency of the inhibition depended on the subunit composition of the OR/ORCO
heteromers, i.e. mainly on the identity of the tuning OR, and specific combinations, such as
AgamOR-7/AgamORCO, were not affected at all (Sato et al. 2008; Nichols et al. 2011; Pask et al.
205
Discussion
2011). In contrast to the amiloride derivatives and similar to DEET, ruthenium red did not appear to
evoke any unspecific effects in calcium imaging experiments. The expected reduction of the Ca2+
levels was not observed, but this might be due to the low ORCO expression in the respective cell
cultures.
The blocker experiments only provide a starting point for further analyses, which should be
performed using the full-length orco sequence and cells showing an improved
transfection/expression rate of the foreign DNA. Since no specific ORCO antagonists, i.e. substances
of the VUAA1 family, were available at the time, such substances were only employed in the
experiments performed on primary M. sexta ORN cell cultures (see 4.3.5).
4.3.4 The loading of M. sexta ORNs with fura-2 AM was not affected
by multidrug resistance transporter blockers in vitro
In the last part of this thesis the function of MsexORCO was investigated in the cells, in which it is
endogenously expressed, in ORNs of M. sexta antennae kept in primary cell culture (Stengl and
Hildebrand 1990). These primary cell cultures contained different types of antennal cells. One type
(type 5 cells) had soma diameters of 4-15 µm and fine uni- or bilateral processes, which could grow
several hundred micrometers in culture. A proportion of 80 % of these cells was recognized by the so
called "olfactory-specific antibody" indicating the identity of ORNs (Stengl and Hildebrand 1990). On
the basis of this morphological criterion it was tried to focus on presumptive ORNs when the ROIs
were chosen for data analysis. In calcium imaging experiments the cells were characterized by a lack
of spontaneous [Ca2+] changes. Indeed, among 987 cells only one cell was detected showing
prominent oscillations of the F340/F380 ratio, i.e. oscillations of the Ca2+ concentration. This was
surprising, because extracellular recordings from different olfactory sensilla revealed spontaneous
spiking activity of ORNs, which often occurred in bursts and was suggested to be also present in
calcium imaging experiments in vitro (Dolzer et al. 2001; Kalinova et al. 2001; Nolte et al. 2013). It
was hypothesized that the uptake of the calcium indicator fura-2 by the cells was too low for the
detection of spontaneous [Ca2+] transients. Manzini and Schild suggested, that ORNs might have
mechanisms to remove xenobiotics and potential cytotoxic substances, probably via multidrug
resistance transporters, since these cells are in direct contact with the environment (Manzini and
Schild 2003). Indeed, blocking experiments suggested the presence of different multidrug resistance
transporters in X. laevis ORNs, which were shown to transport different calcium indicators out of the
cells (Manzini and Schild 2003). In the present experiments it was tested whether fura-2 loading of
presumptive M. sexta ORNs could be improved in the presence of two blockers of multidrug
resistance transporters. In contrast to the experiments performed on X. laevis ORNs, neither
probenecid, which blocks multidrug resistance associated proteins, nor verapamil, which blocks the
P-glycoprotein, changed the kinetics of the fura-2 loading procedure. Since neither spontaneous
[Ca2+] increases nor solvent control-dependent [Ca2+] increases were significantly affected by
probenecid and verapamil, it might be assumed that probenecid- or verapamil-sensitive multidrug
resistance transporters do not play a key role in M. sexta ORNs. In future experiments further
blockers of multidrug resistance transporters may be employed, but so far it appears likely that the
lack of spontaneous activity was not dependent on an active removal of fura-2. Instead a non206
Discussion
optimal condition of the primary cell cultures and depolarization of the ORNs might have been
responsible.
4.3.5 Primary M. sexta ORN cell cultures were not affected by ORCO
modulation
4.3.5.1 ORCO agonists did not significantly activate M. sexta ORNs in vitro
To investigate the function of ORCO in the ORNs of M. sexta the primary cell cultures were
stimulated with different substances of the VUAA1 family, which were all reported to be specific
agonists (VUAA1, OLC12, VUAA4) or antagonists (OLC15) of ORCO and only differed in minor
chemical changes from the VUAA1 structure (Fig. 30, Jones et al. 2011; Chen and Luetje 2012; Taylor
et al. 2012). The agonist OLC12 (also termed VUAA3) was more effective than VUAA1 in the
activation of heterologously expressed, heteromeric OR/ORCO complexes as well as ORCO
homomers of different insect species (D. melanogaster, Culex quinquefasciatus, and Ostrinia
nubilalis, Chen and Luetje 2012). In comparison with VUAA1 (EC50 = 37 µM), VUAA4 exhibited an
approximately tenfold efficiency (EC50 = 2.1 µM) in the activation of heterologously expressed
AgamORCO/OR-65 (Taylor et al. 2012). The hierarchy of these agonists' potencies (VUAA4 > OLC12 >
VUAA1) applied for ORCO orthologues of different insect species (A. gambiae, H. virescens,
H. saltator) and did not depend on the identity of the tuning OR (Taylor et al. 2012). Since VUAA1
was demonstrated to activate MsexORCO in a heterologous expression system (see 4.3.3), it was
expected that MsexORCO is also sensitive to other compounds of this family, probably in the same
order that was demonstrated for other ORCO orthologues. Surprisingly, it was not possible to evoke
statistically significant [Ca2+] increases with any of these compounds. Although VUAA1 activated
heterologously expressed MsexORCO and dose-dependently increased the spontaneous activity as
well as the late long-lasting pheromone response in tip recordings of bombykal-sensitive sensilla of
M. sexta (Fig. 119, Nolte et al. 2013), only a few ORNs showed temporally correlated [Ca2+] increases
after VUAA1 stimulation, whereby the percentage of active cells was not different from solvent
control experiments. Consistent with the low response rate, no concentration-dependent differences
were found. The complete lack of active cells after application of the highest VUAA1 concentration
(500 µM) might have methodical reasons: The highest concentration was always applied at the end
of an experiment, when the condition of the cells and the efficiency of the calcium indicator might
have been weaker than at the beginning. When M. sexta ORNs were stimulated with OLC12, ORCO
was apparently not affected. However, it has to be noted that these experiments were performed
with cell cultures after 65 and 85 days in vitro, respectively, while all other experiments were
performed with cell cultures not older than 34 days. Similar to OLC12, VUAA4 was only employed in a
few experiments, in which the percentages of active cells after VUAA4 stimulation tended to be
lower than after solvent control application, although not statistically significant. One of the reasons
for the lacking efficiency might be the applied concentration. Due to a limited amount of the
compound and its high agonistic potency, VUAA4 was applied exclusively at a concentration of
10 µM, while VUAA1 and OLC12 were employed at a concentration of 100 µM or higher. Thus, in
future experiments higher concentrations might be tested. However, in another study performed on
Chinese hamster ovary (CHO) cells heterologously expressing MsexORCO (stably transfected) both
207
Discussion
agonists OLC12 as well as VUAA4 evoked specific [Ca2+] increases, when applied at the same
concentrations as specified here, with VUAA4 being more potent than OLC12 (Körte 2013).
Therefore, the lack of responsiveness does not appear to be due to a general lack of sensitivity to
these compounds.
4.3.5.2 The ORCO antagonist OLC15 did not affect M. sexta ORNs in calcium
imaging experiments
Interestingly, some chemical modifications of the VUAA1 structure reversed its effect on ORCO from
agonistic to antagonistic. In the case of OLC15 it was sufficient to change the position of the pyridine
nitrogen to extend the ethyl moiety of the phenyl ring to a butyl moiety (Chen and Luetje 2012). This
compound inhibited OLC12- as well as odorant-gated responses of heterologously expressed ORCO
homomers or OR/ORCO heteromers of different insect species (A. gambiae, C. quinquefasciatus,
D. melanogaster, and O. nubilalis). Remarkably, OLC12-gated responses were blocked in a
competitive manner and odorant-gated responses in a non-competitive manner (Chen and Luetje
2012). Consistent with the lack of responsiveness to the ORCO agonists employed, OLC15 was not
capable of activating or inhibiting the basic activity of M. sexta ORNs in calcium imaging experiments
(3.3.2.2).
4.3.5.3 The effects of the amiloride derivatives HMA and MIA appeared to be
ORCO-independent in all cell types tested
Next to the ORCO specific antagonist OLC15 the amiloride derivatives HMA and MIA were also tested
on the primary cell cultures. Exactly as shown for HEK 293 cells, both substances similarly decreased
the F340 and F380 intensity leading to an unspecific increase of the F340/F380 ratio. Apparently, an
increase of the F380 background fluorescence was one of the reasons for the unspecific increase.
Since the effect was demonstrated for HEK 293 cells as well as for all cell types in the primary cell
cultures, it was not only ORCO-independent but also cell type-independent. Interestingly, amiloride
and different derivatives (not HMA and MIA) were taken up by skin cells of Rana pipiens and showed
fluorescence. The maximum fluorescence intensity was achieved by excitation at 360 nm and the
emission maximum was detected at a wavelength of 414 nm. The fura-2 spectrum was only partially
matched since excitation at one of the fura-2 specific excitation wavelengths (340 nm) produced only
weak fluorescence, and no emission was detected at wavelengths higher than 500 nm (Briggman et
al. 1983). However, distinct amiloride derivatives were shown to have slightly different fluorescence
properties. In another study performed on X. laevis ORNs an increased fura-2 fluorescence was
reported once amiloride was present in the bath, which was due to the fluorescence of amiloride and
an uptake into the cells (Lischka and Schild 1993). Therefore, it seems likely that both HMA and MIA
were taken up by the different cell types in the present experiments and exhibited a fluorescence
that impaired the fura-2 measurements. Next to the unspecific increases of the F340/F380 ratio
statistically significant decreases of the F340/F380 ratio were observed, which were slower in their
kinetics. Based on analysis of the single wavelengths these decreases appeared to be [Ca2+]
decreases, but due to two reasons an involvement of ORCO appeared questionable. First, ORCO is
208
Discussion
not the only target of amiloride derivatives. These substances are rather unspecific blockers, which
were originally described as blockers of epithelial Na+ channels, followed by the detection of a broad
blocking spectrum consisting of Na+/H+ antiporters, Na+/Ca2+ exchangers, Na+/K+ ATPases, as well as
different ion channels (Na+-, K+-, Ca2+-channels, nicotinic ACh receptors) and other signaling
components such as different protein kinases (Reviews: Benos 1982; Kleyman and Cragoe 1988).
Concerning olfaction, amiloride was reported to block a Ca2+-activated non-specific cation channel in
A. polyphemus ORNs, which shared high similarities with a non-specific cation channel detected in
M. sexta ORNs (Zufall and Hatt 1991b; Stengl 1993). In addition, amiloride blocked a CNG channel in
frog and rat ORNs (Frings et al. 1992) as well as a Na+-gated non-selective cation channel of the TRP
channel family, which is crucial in lobster olfaction (Bobkov and Ache 2007). Moreover, it blocked
odorant gated currents of heterologously expressed D. melanogaster IRs (Abuin et al. 2011) and
heterologously expressed ORCO orthologues of different insect species (Pask et al. 2013; Röllecke et
al. 2013). Second, the specific ORCO agonists and antagonist did not show effects in most
experiments. Therefore, the observed effects of the unspecific blockers did probably not depend on
MsexORCO. This argument is emphasized by one experiment, in which a single cell showed
prominent [Ca2+] oscillations. The oscillations were not affected by the ORCO agonist VUAA1 but
blocked by both HMA and MIA (Fig. 115), suggesting an ORCO-independent mechanism, such as
inhibition of a Ca2+-activated non-specific cation channel (Zufall and Hatt 1991b).
4.3.5.4 A characterization of ORCO in M. sexta ORNs was not accomplished
In conclusion, it was not possible to perform a characterization of MsexORCO in the primary M. sexta
ORN cell cultures. None of the employed ORCO agonists or antagonists significantly affected the
activity of the ORNs monitored in calcium imaging experiments. The fact that all agonistic substances
(VUAA1, OLC12, VUAA4) were shown to activate MsexORCO in heterologous expression or in situ
studies (Körte 2013; Nolte et al. 2013), suggests that the low responsiveness was not due to a lack of
sensitivity of MsexORCO to these compounds. It is also unlikely, that the reason for the missing
responses was the absence of ORCO in these cells. The expression of ORCO in M. sexta ORNs was
confirmed in antennal slices at the level of mRNA (Grosse-Wilde et al. 2010) and protein (Christian
Flecke, Achim Werckenthin, Karin Großemohr, and Monika Stengl, unpublished). MsexORCO
expression appears to initiate around four days after pupation, increases to an intermediate level
after two days, and highly increases near the end of the pupal period (personal communication with
Prof. Dr. Richard G. Vogt, University of South Carolina, USA). If the expression of ORCO was not
affected by the dissociation of the tissue, all experiments were performed after initiation of ORCO
expression and the majority of experiments even during the intermediate expression or the high
expression state. So far it was not analyzed whether the dissociation procedure or the culture
conditions affect the expression of ORCO. Thus, in future experiments the immunocytochemical
investigation of the primary ORN cell cultures should be top priority. Assuming that the ORCO
expression was not changed in culture, the lack of responses was probably due to a bad condition of
the primary cell cultures, which was also suggested by the lack of spontaneous activity.
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Discussion
4.3.6 The role of ORCO in M. sexta pheromone transduction
The present experiments did not allow for a detailed characterization of MsexORCO. Very low
expression rates in the heterologous system and presumptive bad conditions of the primary M. sexta
ORN cell cultures highly reduced the response rate of the cells and thus complicated the analysis. It
was hypothesized that MsexORCO functions as a pacemaker channel in vivo, which generates SMPOs
and provides a depolarizing inward current that drives the cell to spike threshold. For a precise
analysis of the ion channel properties such as the ion selectivity and I-V relationship, patch clamp
experiments would have been required. Since the majority of HEK 293 cells did not express
MsexORCO and the primary ORN cell cultures did not respond to ORCO modulation, patch clamp
experiments on single cells were not practicable. However, the calcium imaging experiments
demonstrated VUAA1 sensitivity and at least a tendency for cyclic nucleotide sensitivity of
MsexORCO. It was possible to demonstrate that MsexORCO mediates spontaneous as well as VUAA1
gated [Ca2+] increases, suggesting an ion channel function as well as Ca2+ permeability. The findings of
this thesis facilitated the use of the ORCO agonist VUAA1 in long-term tip recordings on pheromone
sensitive sensilla of M. sexta (Nolte et al. 2013, 4.3.6.2).
4.3.6.1 Ionotropic versus metabotropic transduction mechanisms in insect
olfaction
Different studies performed on heterologous expression systems demonstrated that ORCO
heteromerizes with tuning ORs and that the heteromeric complex functions as odorant-gated ion
channel allowing for ionotropic olfactory transduction (Nakagawa et al. 2005; Neuhaus et al. 2005;
Sato et al. 2008; Wicher et al. 2008; German et al. 2013). Since the identity or mutations of the
tuning OR affected the properties of the ion channel, it was suggested that ORCO as well as the
tuning OR contribute to the pore of the channel with unknown stoichiometry (Sato et al. 2008; Jones
et al. 2011; Pask et al. 2011; Nakagawa et al. 2012). From these findings it was concluded for all
insects, that OR/ORCO complexes also mediate odorant-induced ionotropic signal transduction in
vivo (Reviews: Nakagawa and Vosshall 2009; Stengl and Funk 2013; Suh et al. 2014). In addition it
was suggested that metabotropic pathways (cAMP and PKC signaling) are activated via odorant
binding to the tuning OR and serve to modulate the sensitivity of the ionotropic transduction process
in combination with CaM-dependent mechanisms (Wicher et al. 2008; Sargsyan et al. 2011; Getahun
2013; Getahun et al. 2013; Mukunda et al. 2014; Mukunda Shivalingaiah 2014; Review: Wicher
2015).
The OR/ORCO-system is the evolutionary youngest olfactory system of insects (Croset et al. 2010). It
probably evolved in parallel with pterygote insects and thus with insect flight, suggesting its
requirement for the detection of odorants during flight (Missbach et al. 2014; Review: Wicher 2015).
Consistently, OR-expressing ORNs of D. melanogaster were shown to respond faster and with higher
sensitivity than IR- or GR-expressing ORNs to short odorant pulses. Additionally, these ORNs could
resolve repeated stimulations with excitatory odorants up to 5 Hz. Thus, it was suggested, that ORs
are better suited than IRs or GRs to resolve intermittent stimuli in a low concentration, as detected in
air borne odor filaments (Getahun et al. 2012). Other studies demonstrated pulse tracking
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Discussion
frequencies of 3 - 40 Hz for ORNs of different insect species (Rumbo and Kaissling 1989; Marion-Poll
and Tobin 1992; Lemon and Getz 1997; Bau et al. 2002; Bau et al. 2005; Tripathy et al. 2010) and
latencies of 20 - 500 ms (Schneider et al. 1964; de Bruyne et al. 1999; Kaissling 2013). However, the
exact time required for the transduction process (from odorant binding to the receptor until
detection of the transduction current) was difficult to determine since the time required for the
perireceptor events also contributed to those latencies. Previously, astonishing latencies as short as
1.6 ms for Schistocerca americana and pulse tracking frequencies up to 125 Hz for A. mellifera were
shown in EAG recordings (Szyszka et al. 2014). These frequencies might be higher than the pulse
resolution frequencies of the single ORNs because EAGs reflect the summed response of the
recorded cells and according to the volley theory it is likely that single ORNs did not respond to every
stimulus of the pulse train (Wever and Bray 1930, 1937; Szyszka et al. 2014). Additionally, the
importance of these high tracking frequencies is diminished by the fact that the EAGs were
constantly elevated during stimulation and did not return to baseline levels between stimuli,
indicating a physiological state that was shown to prevent a behavioral response (Baker and Haynes
1989; Review: Vickers 2006).
From an evolutionary point of view the OR-based olfactory system of insects needs to be sensitive
and to cover a broad range of odorant concentrations (Stengl and Funk 2013). Therefore it seems
likely that metabotropic receptors are employed, but not ionotropic receptors, which are selected for
speed in the range of microseconds, as shown for hair cells of the auditory system (Corey and
Hudspeth 1979, 1983; Stengl and Funk 2013). Considering an evolutionary relevant behavior such as
sex pheromone detection of M. sexta, the male hawkmoths can distinguish bombykal concentrations
over at least four log units (Dolzer et al. 2003). This range is absolutely required, taking into
consideration that the male moth needs to detect traces of pheromone released by a female located
far away as well as high pheromone concentrations in close proximity or direct contact with the
female, without getting absolutely adapted or "smell-blind" (Stengl 2010; Stengl and Funk 2013).
Given the velocity of the upwind flight of around 3.5 m/s and the wing beat frequency of
approximately 30 Hz, which in combination determine the sampling rate of odorants (Tripathy et al.
2010), there is no need for ionotropic signal transduction in the range of microseconds (Stengl and
Funk 2013). Except for the values reported in the study of Szyszka and colleagues (2014), the kinetics
for olfactory transduction in vivo as well as the shortest latencies (approximately 18 ms) reported for
heterologous expressed ORs (Sato et al. 2008) match the fastest values reported for the latency of
metabotropic signal transduction: Phototransduction in D. melanogaster requires only 20 ms since all
signaling components are tightly packed in signalosomes (Hardie and Raghu 2001). It has to be
determined whether this is the limit or further reduction in reaction time for metabotropic signal
transduction is possible. No need for ORCO in an ionotropic signal transduction process suggests
another function for the ubiquitous coreceptor, most likely as pacemaker channel (Fig. 118, Stengl
2010; Stengl and Funk 2013).
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Discussion
Fig. 118. Hypothetical model for the function of ORCO and pheromone transduction in M. sexta. After entering the
sensillum via pores, pheromones such as bombykal (BAL) are bound by pheromone binding proteins (PBP) as a prerequisite
to pass through the hydrophilic sensillum lymph. Transient binding of bombykal to its presumptive receptor (OR-1) is
probably supported by the sensory neuron membrane protein 1 (SNMP-1) in an unknown fashion. Upon binding of
bombykal a trimeric Gq protein is activated, whose Gαq subunit in turn activates phospholipase Cβ (PLCβ). PLCβ catalyzes the
hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol
2+
(DAG). The increase of IP3 triggers an increase of [Ca ]i via the transient activation of a plasma membrane-bound IP32+
activated Ca channel, lasting only several milliseconds (It). Additionally, IP3 might activate IP3 receptors in the
2+
2+
endoplasmatic reticulum (ER), if not located too far away. The transient influx of Ca activates a Ca -dependent cation
2+
2+
channel (It), which further increases [Ca ]i. In combination with DAG, strong and long [Ca ] rises activate protein kinase C
(PKC), which in turn activates further cation channels (It). Thus, the combined pheromone-gated transduction current (It) is
composed of at least three different ion conductances, triggered by the rapid rise of IP 3. On a longer time scale PKC is also
suggested to phosphorylate ORCO, thereby activating it and/or increasing its cyclic nucleotide sensitivity, whereby it is
presently unknown, whether the pore of the ion channel is formed by one ORCO protein or ORCO multimers. The resulting
2+
conductance is gated metabotropically (Im) and not directly by odorant binding (ionotropically). It affects the cell's [Ca ]
and membrane potential and thus its spontaneous activity, response kinetics, and sensitivity. Question marks indicate
hypotheses without experimental evidence. Figure and legend were modified (redrawn) after (Stengl and Funk 2013).
4.3.6.2 Extracellular tip recordings did not support ionotropic pheromone
transduction
Extracellular tip recordings on pheromone sensitive trichoid sensilla of M. sexta provided an example
for experiments, which do not support an ORCO function in an ionotropic receptor complex (Nolte et
al. 2013). The moth was stimulated with bombykal puffs in intervals of five minutes while VUAA1 was
perfused into the sensillum lymph. Remarkably, VUAA1 did not improve the fast pheromone
response, since the normalized sensillum potential amplitude and the frequency of the first six
bombykal-dependent APs were not increased, and the latency of the first bombykal-dependent AP
was not decreased (Fig. 119). Instead, the spontaneous activity measured before the moth's first
contact with bombykal, as well as the late long-lasting pheromone response (i.e. the background
activity between two bombykal stimulations) were concentration-dependently increased (Fig. 119),
as suggested for a slow pacemaker channel that controls the ORN's calcium concentration and
membrane potential and thereby indirectly also its pheromone sensitivity and response kinetics
(Nolte et al. 2013). When A. aegypti ORCO + OR-8 heteromers were heterologously expressed in
X. laevis oocytes, a highly synergistic effect was detected, when ORCO receptor activator molecule 2
(RAM2, a substance of the VUAA1 family) and the specific odorant (octenol) were simultaneously
212
Discussion
applied (Bohbot and Dickens 2012). Such a synergistic effect, or at least an additive effect, would also
be expected for the simultaneous stimulation of ORCO (via VUAA1) and the bombykal receptor in the
tip recording experiments, but only if ORCO functions as part of an heteromeric OR/ORCO complex,
which mediates ionotropic pheromone transduction in vivo. Since the fast pheromone response was
not improved, the study did not find evidence for an involvement of ORCO in an ionotropic bombykal
transduction cascade (Nolte et al. 2013). Although no adaptation effects to VUAA1 were detected in
the study, the significance of these findings could still be increased, if the presence of the ORCO
agonist would be limited to the pheromone stimulations, instead of being continuously present
during the recording. However, until now it was not possible to find or develop ORCO agonists or
antagonists with sufficient volatility allowing for volatile delivery of these substances and thus actual
simultaneous delivery of ORCO agonist/antagonist and odorant (Jones et al. 2011; Chen and Luetje
2013).
Fig. 119. VUAA1-dependent activation of MsexORCO did not increase the fast but only the late long-lasting bombykal
response and the spontaneous activity of bombykal-sensitive ORNs in situ. A-E. In tip recordings of pheromone-sensitive
trichoid sensilla of M. sexta, which were performed in the moth's activity phase (ZT 1-3) or resting phase (ZT 9-11), VUAA1
was perfused via the recording electrode into the sensillum lymph. Every 300 s the moth was stimulated with bombykal
(1 µg dissolved in 10 µl n-hexane). The effects of different concentrations of VUAA1 and the solvent control (0.1 % DMSO,
Ctr) were compared with each other for the normalized sensillum potential amplitude (norm. SPA, A), the AP frequency (B),
the latency of the first bombykal-dependent AP (C), the spontaneous activity measured before the moth's first contact with
bombykal (D), as well as the late long-lasting pheromone response (E, n.s. = not significant, * p < 0.05, ** p < 0.01, *** p <
0.001, Mann-Whitney test). The figure was modified from (Nolte et al. 2013). VUAA1 did not increase the normalized
sensillum potential amplitude (norm. SPA, A), the AP frequency (B), nor it decreased the latency of the first bombykaldependent AP (C), as suggested for enhanced ionotropic pheromone transduction. Instead the spontaneous activity
measured before the moth's first contact with bombykal (D), as well as the late long-lasting pheromone response (E) were
concentration-dependently increased, as suggested for a pacemaker channel.
213
Discussion
4.3.6.3 Does ORCO function as pacemaker channel?
The finding that injection of 8-Br-cAMP into the sensillum lymph did not increase the background
activity of the ab3A neuron in D. melanogaster, although DmelORCO was activated by cAMP, argued
against a pacemaker function of ORCO in the fruitfly (Getahun et al. 2013). On the other hand
injection of VUAA1 into the same sensillum significantly elevated the spike frequency of the ab3A
neuron, arguing again for the pacemaker function (Su et al. 2012). The detection of three orco
candidate genes in the firebrat Thermobia domestica, which does not express any conventional ORs
(Missbach et al. 2014), indicated that ORCO is not part of an ionotropic OR complex and fulfills a
different function in this species, possibly as pacemaker. Taking into consideration that ORCO ion
channel complexes are spontaneously active (Sato et al. 2008; Wicher et al. 2008; Jones et al. 2011;
Sargsyan et al. 2011; Nolte et al. 2013; Mukunda et al. 2014), it seems awkward to provide the
transduction current of a sensory system by a leaky ion channel, arguing against the ionotropic
receptor function. Next to DmelOR/ORCO heteromers, DmelORCO homomers were detected in vitro
(Neuhaus et al. 2005; German et al. 2013) and in vivo (Benton et al. 2006). In different studies the
homomers were shown to have slightly different physiological properties than the heteromers (Jones
et al. 2011; Pask et al. 2011; Bohbot and Dickens 2012; Nolte et al. 2013; Pask et al. 2013; Mukunda
et al. 2014). Therefore, it is conceivable that ORCO homomers fulfill a different function in vivo than
OR/ORCO heteromers, possibly as pacemaker.
How can evidence for one or the other function of ORCO be provided? All studies published so far
failed to clearly falsify one of the possible ORCO functions. Since the discovery of ORCO, only a few
studies were performed aiming at the characterization of its function in vivo and the investigation of
the olfactory signal transduction cascade in vivo (Yao and Carlson 2010; Getahun et al. 2013; Nolte et
al. 2013). Studies showing ionotropic ORCO-dependent signal transduction in vitro may not prove,
that this transduction cascade also plays a crucial role in vivo. The OR/ORCO complex might be in a
complete different state in vivo. On the other hand, studies claiming ORCO-independent
metabotropic olfactory transduction cannot provide clear evidence that the observed second
messenger pathways are involved in the transduction cascade instead of playing an important role in
the modulation thereof. Odorant-induced metabotropic signal transduction and a function of ORCO
as pacemaker channel can only be falsified in vivo via elimination of G protein signaling in the
respective ORNs. Since different metabotropic cascades might substitute for each other, if only one
component is eliminated, a complete elimination is required. It cannot be ensured that different
G protein blockers, which were described for mammalian systems, provide identical effects in
insects. Additionally, it cannot be ensured that the blockers reach their target molecules, because in
vivo preparations usually are not as accessible as cells in culture. Therefore, a genetic knockout
appears to be more promising than the use of pharmacological blockers. To prevent any
developmental effects, the knockout must not be constitutive but needs to be inducible. Taking all
this into consideration, D. melanogaster mutants with a temperature-inducible, ORN specific knockout of both Gγ subunits, which are present in fruit flies (Ray and Ganguly 1992; Schulz et al. 1999;
Boto et al. 2010), could be employed. In contrast, odorant induced ionotropic signal transduction and
thus an ORCO function as part of an ionotropic OR complex can only be falsified by the
demonstration of proper olfactory signaling in the absence of ORCO in vivo. Since ORCO's "chaperon
function" is absolutely required for the functional membrane insertion of conventional ORs in vivo
(Larsson et al. 2004; Benton et al. 2006), the ultimate challenge appears to be the generation of an
ORCO mutant, which ensures functional trafficking of ORs but completely lacks ion channel function.
214
Discussion
Different ORCO mutants were designed, but none of them unrestrictedly fulfilled those requirements
(Wicher et al. 2008; Nakagawa et al. 2012; Kumar et al. 2013; Turner et al. 2014). Surely, the ORCO
PKC mutant (Sargsyan et al. 2011) is a large step in this direction, since OR trafficking was unaffected,
while the PKC and cAMP sensitivity and thus metabotropic activation of ORCO was abolished in this
mutant. In contrast to WT ORCO, the ORCO PKC mutant did not provide a resting current different
from non-transfected control cells, indicating that the spontaneous activity is also abolished
(Sargsyan et al. 2011). However, it was concluded that this mutant still provides ORCO-dependent
ionotropic signaling and, thus, does not completely lack ion channel function (Sargsyan et al. 2011;
Getahun et al. 2013). In this respect it would be interesting to know, whether the ORCO PKC mutant
is activated by VUAA1. In addition, it would be absolutely essential to provide direct evidence for
G protein coupling of insect ORs. Several studies suggested that heterologous expressed insect ORs
can couple to coexpressed G proteins (Wetzel et al. 2001; Sakurai et al. 2004; Nakagawa et al. 2005;
Grosse-Wilde et al. 2007; Deng et al. 2011; Pregitzer et al. 2014), but the direct interaction of ORs
and G proteins has not been demonstrated so far.
4.4 A comparison between central AME and peripheral ORN
pacemaker neurons
The present thesis aimed at further characterization and comparison of two types of pacemaker
neurons: central AME pacemaker neurons of the cockroach R. maderae (Petri and Stengl 1999),
which control daily behavioral rhythms of the animal, and peripheral ORN pacemaker neurons of the
hawkmoth M. sexta (Stengl and Hildebrand 1990), which are assumed to control daily rhythms of
olfactory sensitivity (i.e. their own sensitivity). Both cell types were shown to express clock genes:
AME neurons and ORNs show PER immunoreactivity (Schuckel et al. 2007; Werckenthin 2013) and
AME neurons express oscillating mRNA levels of per, tim1, and cry2 (Werckenthin et al. 2012).
Circadian pacemaker neurons are usually defined as neurons, which express clock genes and are part
of the circadian pacemaker system that drives a rhythmic output. From an electrophysiological
standpoint pacemaker neurons are defined as neurons with the intrinsic capability to generate
subthreshold membrane potential oscillations, which lead to the generation of APs, preferably in
bursts, without the need of extrinsic inputs, if the threshold is exceeded (Review: Ramirez et al.
2004). However, by definition this intrinsic capability is not inevitably claimed for circadian
pacemakers. It arises from an interplay of different ion channels. Pacemaker channels provide a
depolarizing inward current that drives the resting membrane potential to the threshold of other ion
channels such as voltage-activated Na+ channels, whose opening initiates APs. The depolarization in
turn activates other voltage activated channels such as Kdr channels, which re- and eventually
hyperpolarize the neuron.
Calcium imaging experiments performed on single AME neurons suggested a model, in which low
voltage-activated Ca2+ channels as well as HCN channels but not slowly inactivating Na+ channels
provide the pacemaker current. Increasing voltage during APs activates high voltage-activated Ca2+
channels that mediate prominent [Ca2+] transients. The combination of increasing voltage and Ca2+
influx then activates SK channels and voltage activated K+ channels, which repolarize the cell and
restrain the activity (see 1.4.10, Fig. 15, Wei and Stengl 2012). In the present patch clamp
215
Discussion
experiments with dissociated AME neurons different ion channels (Ca2+, K+, Na+, and probably also Cland non-specific cation channels such as HCN channels) were electrophysiologically demonstrated
for the first time. However, a detailed pharmacological analysis was not possible because it was
difficult to obtain stable recordings allowing for the application of different pharmacological
substances (see 4.2).
The ORNs of M. sexta have been electrophysiologically far better investigated than the AME neurons
of R. maderae and several ion channels have been characterized (Zufall et al. 1991; Stengl et al.
1992b; Stengl 1993, 1994; Dolzer 2002; Dolzer et al. 2008; Krannich 2008; Krannich and Stengl 2008;
Review: Stengl 2010). Different studies suggested a model, in which MsexORCO provides the
pacemaker current that depolarizes the neuron. Thereby rapidly inactivating voltage-activated K+
channels (IA) as well as slowly inactivating Kdr channels are activated. Additionally, the ORCOdependent influx of Ca2+ activates BK channels as well as Ca2+ dependent Cl- channels. The interplay
of these ion channels is suggested to generate subthreshold membrane potential oscillations, which
might elicit APs, if the threshold for voltage-activated Na+ channels is exceeded. Increases of the
cAMP level (probably by stimulation of octopamine receptors) or activation of PKC (probably by
strong pheromone stimuli) are assumed to increase the open probability of ORCO and thereby
increase amplitude and/or frequency of the subthreshold membrane potential oscillations. Rapidly
inactivating L-type Ca2+ channels and slowly inactivating CNG channels might also be activated via
cAMP, allowing for additional Ca2+ influx, which mediates negative feedback (Stengl 2010). Next to
ORCO, HCN channels were also detected in 23 % of the M. sexta ORNs in cell culture, which were
activated by 8-Br-cAMP and 8-Br-cGMP and conducted a current (Ih) at hyperpolarized potentials
peaking at -80 mV (Krannich 2008). Therefore, it seems likely that in a subpopulation of ORNs the
pacemaker current can be provided by ORCO and HCN channels. The experiments described in 3.3.1
and 3.3.2 support the assumption that MsexORCO functions as leaky ion channel, which mediates
spontaneous Ca2+ influx and is activated by cAMP. Due to difficulties with the heterologous
expression of MsexORCO, which were most probably caused by missing amino acids in the
N-terminal tail, and the condition of the primary ORN cell cultures a detailed electrophysiological
characterization of the channel properties was not possible.
216
Appendix
5 Appendix
5.1 Manduca sexta rearing
Artificial diet (as used at the Max Planck Institute for Chemical Ecology, Jena):




















1.8 l water
144 g wheat germ
140 g corn meal
76 g soy flour
75 g casein
60 ml 37 % formalin
46 g agar
36 g sugar
24 g salt
12 g ascorbic acid
9 ml linseed oil
6 g sorbic acid
5 g cholesterol
3 g methylparaben
30 mg nicotinic acid
15 mg riboflavin
7 mg folic acid
7 mg pyridoxine
7 mg thiamine
0.6 mg biotin
Artificial diet (as used at the University of Kassel):





1.8 l water
500 g flour mix
32 ml vitamin mix
85 g agar
4.5 ml rapeseed oil
217
Appendix
Flour mix (1000 g):









312.5 g soy flour
288.8 g rye flour
288.8 wheat flour
77 g casein
29 g ascorbic acid
9.6 g methylparaben
9.6 g sorbic acid
2.9 g CaCO3
2.9 g NaCl
Vitamin mix (100 ml):







100 ml water
100.3 mg nicotinic acid
50.2 mg riboflavin
23.4 mg folic acid
23.4 mg pyridoxine
23.4 mg thiamine
2.0 mg biotin
Sugar solution for artificial flowers:




Water
187 mM glucose
154 mM fructose
464 mM saccharose
218
Appendix
5.2 Primer sequences
Tab. 20. Primer sequences
Primer name
Primer sequence
MsexOrco BamHI for
cgc gga tcc ATG ACC ATG CTT CTG CGG AA
MsexOrco EcoRI rev
ccg gaa ttc CTA TTT CAG CTG CAC CAA C
MsexOr-1 KpnI for
cc ggt acc ATG ATA TTT ATG GAC GAT CCT CTA TCA AAG
MsexOr-1 XhoI rev
ga ctc gag TTA GTT AGA AAC GGT GCG AAG AAA TG
MsexOr-4 KpnI for
cc ggt acc ATG AAG TTT TTT GTA GAC GGC AGC GAA ATA
MsexOr-4 XhoI rev
ga ctc gag TTA GCT CTC ATC TTT GGC GAT TGT TTG A
MsexSnmp-1 for
ATG CGG CTG GCA AGG GGA ATT AAG
MsexSnmp-1 rev
TTA CAT GTT GAT TTT TGG AGG CTC ATG AC
The table lists the primer sequences used for amplification of the respective genes. Coding sequences are shown in
capitals. In cases where a restriction site was induced, the respective sequence and the appropriate endonuclease is
indicated in bold. for: forward primer, rev: reverse primer, BamHI: Bacillus amyloliquefaciens H1, EcoRI: Escherichia coli R1,
KpnI: Klebsiella pneumoniae I, XhoI: Xanthomonas holcicola I. Modified after (Nolte et al. 2013).
5.3 Composition of solutions
5.3.1 Solutions for primary AME cell cultures
Dissection medium / washing medium 1 (200 ml):





178 ml Leibovitz´s L-15 medium without L-glutamine (L-15, PAA, part of GE Healthcare,
Cölbe, Germany)
20 ml heat-inactivated fetal bovine serum (FBS Gold, PAA)
2 ml penicillin (10000 units/ml)/streptomycin (10 mg/ml)-mixture (Pen/Strep, Sigma-Aldrich,
Taufkirchen, Germany)
pH-adjustment to 7.0
osmolarity-adjustment to 380 mosm/l with D-mannitol
219
Appendix
Supplemented Hank's Balanced Salt Solution (sHBSS, 100 ml):






15 ml 10 x HBSS (Gibco, Life Technologies, Carlsbad, CA, USA)
1 ml Pen/Strep (Sigma-Aldrich)
15 mg phenylthiourea (PTU, Sigma-Aldrich)
2 mg phenol red
84 ml ddH2O
Dissociation medium 1 (5 ml):



5 ml sHBSS
5 mg papain (crude powder, Sigma-Aldrich)
optionally: 1 mg L-cysteine (Sigma-Aldrich)
Dissociation medium 2 (10 ml):



10 ml sHBSS
10 mg collagenase (C2674, Sigma-Aldrich)
40 mg dispase (D4693, Sigma-Aldrich)
Washing medium 2 (100 ml):



100 ml L-15 without L-glutamine (PAA)
280 mg yeastolate
280 mg lactalbumin
Culture medium 1 (200 ml):





10 ml heat-inactivated FBS Gold (PAA)
2 ml L-glutamine (Gibco, Life Technologies, part of Thermo Fisher Scientific)
osmolarity-adjustment to 380 mosm/l with D-mannitol
Culture medium 2 (100 ml):






1 ml supplemental mix
1 ml L-glutamine (Gibco)
100 µl gentamicin (Invitrogen, Life Technologies)
10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
pH-adjustment to 7.0, osmolarity-adjustment to 380 mosm/l with D-mannitol
220
Appendix
Supplemental mix (10 ml):





2 g glucose
0,8 g fructose
0,35 g L-proline
0,06 g imidazole
10 ml L-15 (PAA)
5.3.2 Solutions for primary M. sexta ORN cell cultures and the
MRRL-CH1 cell line
Recipes for supplemented Hank's Balanced Salt Solution (HBSS), dissociation medium 1, and
dissociation medium 2 are identical to the recipes described in 5.3.1.
2:1 medium (30 ml):


20 ml L-15 with 5 % FBS (culture medium 1, see 5.3.1)
10 ml conditioned medium (supernatant of the MRRL-CH1 cell line, see 2.3.5)
Cell line nutritive medium (CLNM, 550 ml):


500 ml Grace's Insect Medium (1x), supplemented with L-glutamine, lactalbumin, and
yeastolate (Gibco)
50 ml heat-inactivated FBS Gold (PAA)
5.3.3 Solutions for HEK 293 cell culture
Culture Medium (500 ml):


450 ml Dulbecco’s Modified Eagle Medium/Ham’s F-12 medium with L-glutamine
(DMEM/Ham’s F-12, PAA)
50 ml heat-inactivated FBS (PAA)
Separation medium (5 ml):


4.5 ml HBSS (PAA)
0.5 ml Trypsin (5 g/l)-EDTA (2 g/l) solution (Sigma-Aldrich)
221
Appendix
5.3.4 Solutions for immunocytochemistry
10 x Phosphate-buffered saline (PBS, 100 ml):






1.37 M NaCl
27 mM KCl
100 mM Na2HPO4
18 mM KH2PO4
ddH2O ad 100 ml
Fixation buffer (100 ml):





10 ml 10x PBS
70 ml ddH2O
4 g paraformaldehyde (Sigma-Aldrich)
ddH2O ad 100 ml
Wheat germ agglutinin solution (WGA, 10 ml):


250 µl Texas Red®-X-conjugated WGA solution (1 mg/ml, Invitrogen)
7.75 ml HBSS (PAA)
Blocking buffer (20 ml):



2 ml 10x PBS
20 µl Triton™ X-100 (Sigma-Aldrich)
1 ml normal goat serum (NGS, Invitrogen)
222
Appendix
5.3.5 Solutions for electrophysiology and calcium imaging
Tab. 21. Extracellular ringer solutions for insect neurons (6 mM CaCl2)
NaCl
TEA-Cl
KCl
CaCl2
NiCl2
EGTA
HEPES
Glucose
Standard
156
TEA
136
20
4
6
4
6
10
5
Low Ca
156
2+
High Ca
114
2+
NiCl2/CaCl2
156
NiCl2
156
4
4
20
4
2
4
4
10
5
10
5
(1)
10
5
10
5
10
5
6
The table summarizes the constituents (in mM) of different extracellular solutions, which are based on the standard
extracellular saline containing 6 mM CaCl2. Further extracellular solutions were prepared by adding additional substances
to the standard extracellular saline without changing the concentrations of the remaining constituents. Occasionally the
pH indicator phenol red was added at a concentration of 10 mg/l. The pH was adjusted to 7.1 and the osmolarity to
380 mosm/l, using mannitol.
Tab. 22. Extracellular ringer solutions for insect neurons (1 mM CaCl2)
NaCl
TEA-Cl
KCl
CaCl2
NiCl2
HEPES
Glucose
Standard
156
4
1
TEA
136
20
4
1
10
5
10
5
NiCl2
156
4
1
10
5
The table summarizes the constituents (in mM) of different
extracellular solutions, which are based on the standard
extracellular saline containing 6 mM CaCl2. Further
extracellular solutions were prepared by adding additional
substances to the standard extracellular saline without
changing the concentrations of the remaining constituents.
Occasionally the pH indicator phenol red was added at a
concentration of 10 mg/l. The pH was adjusted to 7.1 and
the osmolarity to 380 mosm/l, using mannitol.
Tab. 23. Intracellular solutions for insect neurons
KCl
CsCl
CaCl2
EGTA
HEPES
Glucose
Standard
160
1
11
10
5
Cs
160
1
11
10
5
The table summarizes the constituents (in mM) of
intracellular solutions, which are based on KCl or CsCl. The
pH was adjusted to 7.1 and the osmolarity to 340 mosm/l,
using mannitol.
Tab. 24. Extracellular ringer solutions for HEK 293 cells and SF9 cells
NaCl
KCl
CaCl2
MgCl2
HEPES
PIPES
Glucose
Probenecid
HEK 293
135
5
1
1
10
10
SF 9
12
21
3
18
The table summarizes the constituents (in mM) of
extracellular ringer solutions used for calcium imaging
experiments on HEK 293 and SF9 cells. The pH was adjusted
to 7.4 (HEK 293) or 7.2 (SF9). No further adjustment of
osmolarity was performed.
10
170
1
223
Appendix
5.4 Electrophysiological characterization of AME neurons at
the network level
Fig. 120. Dissection and application times. A-H. The figure shows the temporal distribution of AME-dissections (A, C, E, G)
and PDF-, 8-Br-cAMP-, 8-Br-cGMP-, or 8-pCPT-2'-O-Me-cAMP (007) applications (B, D, F, G) during extracellular long-term
recordings. An accumulation of dissections between Zeitgeber time (ZT) 4 and ZT 11 and applications between ZT 6 and
ZT 13 can be seen for PDF, 8-Br-cAMP, and 8-Br-cGMP.
224
Appendix
Fig. 121. AME neurons burst with regular interburst-intervals. A-D. Extracellular recording from an isolated AME. A.
Original recording trace, showing summed action potentials (SAPs) as upward deflections from the baseline. The grey bar
marks the position of the detail, which is magnified in D. B. The corresponding mean frequency lay around 7.5 Hz and
slightly decreased. C. The corresponding instantaneous frequency (1/interevent-intervals) showed a narrow band around
3 Hz and a cloud of points lying above. D. The detail of the recording trace illustrates that the recorded neurons were
generating short bursts with regular interburst-intervals around 0.35 s, causing the instantaneous frequency band around
3 Hz (C).
225
Appendix
Fig. 122. PDF inhibits or activates AME neurons and promotes bursting. A-E. Extracellular recording from an AME explant.
A. The recording trace shows positive and negative summed action potentials (SAPs) originating from different AME
neurons. The arrows indicate the application of PDF in the vicinity to the tissue (0.3 pmol, 0.45 pmol, and 1.5 pmol). B, C.
The AME neurons generating positive SAPs were first activated by PDF as indicated by the corresponding mean frequency
(B) and the increasing density of the instantaneous frequency band (C). Simultaneously, the cells started to burst, indicated
by the upward deflections. Apparently, further PDF applications inhibited the electrical activity of these cells. D, E. Another
group of AME neurons, generating negative SAPs, was dose-dependently inhibited by PDF. After the first application of
PDF the activity slowly decreased and oscillated, as indicated by the noise of the instantaneous frequency band. After the
second application the activity was almost completely abolished. The data were re-analyzed from (Funk 2005).
226
Appendix
Fig. 123. Application of 8-Br-cAMP transiently activates AME neurons. Original recording trace of an extracellular
recording from an isolated AME in the presence of the Cl channel antagonist picrotoxin (PTX, 0.5 mM). Summed action
potentials (SAPs) from the recorded neurons can be seen as upward deflections from the baseline. The arrows indicate the
application of each 200 pmol 8-Br-cAMP (via Picospritzer). 8-Br-cAMP reproducibly and transiently elicited SAPs with
higher amplitude.
5.5 Patch clamp analysis of single AME neurons
Fig. 124. Outward currents reaching a plateau were regarded as caused by the superposition of a weak sustained inward
current. A-C. Whole-cell patch clamp recording from an AME neuron in a primary cell culture performed in saline
containing 1 mM CaCl2. A. Voltage step protocol: The cell was kept at a holding potential of -60 mV and stimulated with
depolarizing voltage steps from -140 mV to +80 mV to activate voltage-dependent currents. B. Current response of the cell
showing non-inactivating outward currents (IK, arrow 1), tail currents (Itail, arrow 2), the first (INa, arrow 3), and the second
transient inward current (ICa, arrow 4). The current traces corresponding to the highest voltage steps converged. C. I-V
relationship of the sustained whole-cell current, measured at the position indicated by arrow 1. The outward current
activated at -50 mV and reached a plateau at +30 mV, probably due to an counteracting, non-inactivating inward current
2+
+
(Iin,sust) or the presence of Ca -activated K currents. The inlay shows a photograph of the recorded neuron. Scale bar:
20 µm.
227
Appendix
Fig. 125. Perfusion with control solution did not affect whole-cell current components. A-D. Whole-cell patch clamp
recording from an AME neuron in a primary cell culture performed in saline containing 1 mM CaCl2. A, C. I-V relationships
for the sustained outward current (IK, A) and for the transient currents (C, outward current component = IK,trans, inward
current component = ICa), measured before (black squares) or after application of control solution (open squares). B. Time
course of the area under the I-V curves (AUC) for IK for all protocol runs. D. Time course of the peak value of ICa. Both time
courses illustrate that the current decrease happened spontaneously and apparently was not affected by the application of
control solution (arrows). Data for the I-V relationships are given as means ± SEM (in each case n=3 protocol runs). For
more information see Fig. 67.
228
Appendix
Fig. 126. INa is blocked by PDF and TTX. A-J. Whole-cell patch clamp recording from an AME neuron in a primary cell culture
performed in saline containing 1 mM CaCl2. A. Voltage step protocol used to activate voltage-dependent currents. B, C, E,
F. Representative current traces under control conditions (B), after perfusion (2 min) with pigment-dispersing factor (PDF,
500 nM, C), after perfusion (2 min) with the sodium channel blocker tetrodotoxin (TTX, 10 nM, E), and after further
incubation (5 min) in TTX (F). A reduction of the transient outward current (IK,trans, arrow 1), the sustained IK (arrow 2), the
tail currents (Itail, arrow 3), the first (INa, arrow 4), and the second transient inward current ICa (arrow 5) can be seen. F. After
long incubation in TTX a sustained inward current component (Iin,sust, arrow 6) was activated. D. Photograph of the recorded
neuron. Scale bar: 20 µm. G-J. I-V relationships for the sustained currents (G, measured at the position indicated by arrow 2
in B), for the transient currents (H, arrow 1), for the mean Itail (I, calculated over the range indicated by bar i), and for the
negative peak of INa (J, bar j). PDF application resulted in a strong reduction of sustained (IK, G), transient (IK,trans, H), and Itail
outward current components (I), as well as the fast INa (J, also shown in (Wei et al. 2014)) and the slower ICa (inward current
component in H). After washout of PDF with saline containing TTX IK (G), IK,trans (H), Itail (I), and ICa (H) further decreased,
probably in a TTX-independent manner. J. The residual INa was almost completely blocked by TTX. G, I. After long
incubation in TTX Iin,sust developed and counteracted IK (G) and Itail (I) outward currents resulting in the downward bend of
the respective I-V curves at voltages higher than +60 mV. Data for the I-V relationships are given as means ± SEM (n = 3
protocol runs before treatment, n = 7 after PDF application, n = 4 after TTX application, n = 4 after long incubation in TTX).
229
Appendix
Tab. 25. Effects of TEA application
A. 6 mM CaCl2
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=5
100.0 %
n=5
IK,trans
n=4 100.0 %
n=4
INa
n=1
n=1
n=1
33.3 %
33.3 %
33.3 %
ICa
n=2
n=1
n=3
n=3
Iin,sust
66.7 %
n=1
20.0 %
33.3 %
n=1
n=3
n=5
20.0 %
60.0 %
80.0 %
Iin,sust
n=6
n=2
B. 1 mM CaCl2
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=8
n=2
80.0 %
20.0 %
n=10
IK,trans
n=8
n=1
n=1
INa
ICa
n=4
80.0 %
10.0 %
10.0 %
n=10
n=4
n=3
57.1 %
42.9 %
n=1
n=5
n=7
20.0 %
n=2
n=10
60.0 %
20.0 %
20.0 %
In five whole-cell patch clamp recordings performed in 6 mM and ten recordings performed in 1 mM CaCl2
tetraethylammonium (TEA) was bath-applied. Effects on different whole-cell current components, such as the sustained
potassium outward current IK, the transient outward current IK,trans, the transient inward currents INa and ICa, and the
it.
Tab. 26. Effects of 4-AP application
A. 6 mM CaCl2
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=3
n=2
37.5 %
25.0 %
n=3
37.5 %
n=8
IK,trans
n=3
37.5 %
n=1
12.5 %
n=3
n=1
n=8
37.5 %
12.5 %
INa
n=1
n=2
n=2
n=1
n=6
16.7 %
33.3 %
33.3 %
16.7 %
ICa
n=2
n=1
n=1
n=3
28.6 %
14.3 %
14.3 %
42.9 %
n=7
Iin,sust
n=1
16.7 %
n=1
n=4
16.7 %
66.7 %
n=6
B. 1 mM CaCl2
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=1
20.0 %
n=2
n=2
40.0 %
40.0 %
n=5
IK,trans
n=2
n=1
n=1
n=1
40.0 %
20.0 %
20.0 %
20.0 %
n=5
INa
n=2
50.0 %
n=2
50.0 %
n=4
+
ICa
n=2
n=1
n=1
n=1
n=5
40.0 %
20.0 %
20.0 %
20.0 %
Iin,sust
n=2
50.0 %
n=1
n=1
n=4
25.0 %
25.0 %
The K channel blocker 4-aminopyridine (4-AP, 1mM or 4 mM) was bath-applied in five whole-cell patch clamp recordings
(6 mM CaCl2) and ten recordings (1 mM CaCl2), respectively. Effects on different whole-cell current components, such as
the sustained potassium outward current IK, the transient outward current IK,trans, the transient inward currents INa and ICa,
and the sustained inward current Iin,sust were summarized. Percentages relate to the number of cells (# cells) expressing the
respective current component. Uncorrelated decrease: decrease, which was not caused by the application.
230
Appendix
Tab. 27. Effects of PDF application
A. 6 mM CaCl2
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=1
n=1
n=6
n=1
n=9
INa
n=1
n=1
n=3
n=2
n=1
n=8
12.5 %
12.5 %
37.5 %
25.0 %
12.5 %
INa
n=4
n=1
n=17
n=2
n=3
n=27
14.8 %
3.7 %
63.0 %
7.4 %
11.1 %
11.1 %
11.1 %
66.7 %
11.1 %
ICa
n=1
n=1
33.3 %
33.3 %
Iin,sust
n=1
33.3 %
n=3
n=1
n=1
n=6
12.5 %
12.5 %
75.0 %
n=8
B. 1 mM CaCl2
Decrease
Increase
No effect
Not analyzable
# cells
IK
n=9
n=2
n=7
n=16
n=3
n=37
24.3 %
5.4 %
18.9 %
43.2 %
8.1 %
ICa
n=8
n=3
n=6
n=7
n=3
n=27
29.6 %
11.1 %
22.2 %
25.9 %
11.1 %
Iin,sust
n=7
n=2
n=8
n=10
n=3
n=30
23.3 %
6.7 %
26.7 %
33.3 %
10.0 %
The neuropeptide pigment-dispersing factor (PDF) was bath-applied in nine whole-cell patch clamp recordings (6 mM
CaCl2) and 37 recordings (1 mM CaCl2), respectively. PDF was applied via pipette (1 - 250 pmol), Picospritzer (1 fmol - 1
pmol), or the perfusion system (500 nM). Effects on different whole-cell current components, such as the sustained
potassium outward current IK, the transient inward currents INa and ICa, and the sustained inward current Iin,sust were
summarized. Percentages relate to the number of cells (# cells) expressing the respective current component.
5.6 Immunocytochemical characterization of heterologously
expressed MsexORCO
Tab. 28. Statistics of immunocytochemical data 1
Fig.
Fig.
84
Fig.
84
Data group
orco (or-1/4, snmp-1) Rotifect
orco (or-1/4, snmp-1) X-tremeGENE
n
28
7
K2
3.173
-
p (norm.)
0.205
-
Mean (%)
6.939
7.335
SD (%)
3.697
3.780
SEM (%)
0.699
1.429
orco
orco + or-1
orco + or-1/4 +snmp-1
18
7
10
4.386
0.032
0.112
0.984
6.735
5.019
8.927
3.486
3.250
3.648
0.822
1.228
1.154
The K2 and p value computed by the D'Agostino-Pearson normality test as well as descriptive statistical parameters such
as mean, standard deviation (SD), and standard error of the mean (SEM) are shown. For further information see Fig. 84.
231
Appendix
Tab. 29. Statistics of immunocytochemical data 2
Fig.
Data groups
Fig.
84
Fig.
84
orco (or-1/4, snmp-1) Rotifect
orco
orco + or-1
orco + or-1/4 +snmp-1
p value
(ANOVA)
-
Pairwise comparison
orco (or-1/4, snmp-1) Rotifect vs.
0.0823
Pairwise
test
UTT
p value
-
-
-
0.8020
P values for groupwise comparisons (ANOVA) or pairwise comparisons (unpaired t-test, UTT) are shown. For further
information see Fig. 84.
5.7 Calcium imaging experiments on heterologous
expression systems
Fig. 127. SF9 cells do not respond significantly to forskolin. A. Normalized calcium imaging data for 114 SF9 cells
transfected with MsexOr-4. Each line represents the percentage deviation of the fluorescence ratio from the mean of the
-5
first ten values (% Δ(F340/F380)) for one cell. After application of 10 M forskolin (FSK, 100 µl, arrow), three cells showed
2+
threshold-exceeding [Ca ] increases with different kinetics. B. The percentages of active cells, transfected with MsexOr-1
-5
or MsexOr-4, after application of 10 M FSK or 0.1 % DMSO were compared (n.s. = not significant, Wilcoxon signed rank
test, n = number of experiments).
Tab. 30. Statistics of calcium imaging data (heterologous expression system) 1
Fig.
Data group
Fig.
87
Fig.
88A
Fig.
88B
Fig.
89A
Median
(%)
0.2388
0.199
< 0.0001
0.1847
-
25th
percentile
(%)
7.103
80.28
7.539
81.43
0.0
12.32
13.25
58.57
23.08
84.38
22.22
89.38
0.0
70.80
78.15
35.29
75th
percentile
(%)
71.74
90.69
70.97
92.49
37.5
88.24
90.48
89.84
0.3612
0.3087
< 0.0001
1.087
1.754
2.624
4.651
4.082
3.781
7.060
9.091
5.683
n
K2
p (norm.)
ATP 10-6 M
ATP 10-5 M
ATP 10-4 M
ATP 10-3 M
ATP (low concentration)
ATP (high concentration)
DMSO
BSA 10-5 M
BSA 10-4 M
BSA 10-3 M
BSA, transfected cells
BSA, non-transfected cells
1
16
4
2
17
6
157
1
2
10
6
7
2.864
3.226
173.5
3.378
-
BSA(-) 10-6 M
BSA(-) 10-5 M
BSA(-) 10-4 M
9
15
16
2.037
2.351
19.27
232
Appendix
Fig.
89B
Fig.
90A
Fig.
90B
Fig.
90C
Fig.
90D
Fig.
90E
Fig.
90F
Fig.
91
Fig.
92C
Fig.
92D
Fig.
92E
Fig.
93
Fig.
94
Fig.
95
Fig.
96
Fig.
97A
Fig.
BSA
DMSO
BSA(-)
DMSO
non-transfected
orco + or-1
orco + or-1 + DmelSnmp-1
orco + or-1 + MsexSnmp-1
non-transfected
orco + or-1 + snmp-1
13
69
40
20
18
6
23
4
5
6
24
25
14
11
5.015
71.50
17.05
13.67
22.29
21.02
11.25
15.33
8.129
18.42
0.0815
< 0.0001
0.0002
0.0011
< 0.0001
< 0.0001
0.0036
0.0005
0.0172
0.0001
13.47
0.0
2.199
0.305
0.0
0.0
2.273
0.0
0.0
0.0
0.248
0.50
2.275
0.0
77.50
0.0
4.146
3.385
0.0
0.0
3.509
0.0
0.0
0.0
1.241
1.0
4.0
2.381
88.99
1.946
6.812
7.081
2.0
1.136
5.263
4.592
6.769
0.248
4.0
3.50
9.342
5.0
non-transfected (Ringer)
non-transfected (+ DMSO)
18
6
22.29
-
< 0.0001
-
0.0
0.0
0.0
0.0
2.0
0.248
orco + or-1 + snmp-1 (Ringer)
orco + or-1 + snmp-1 (+ DMSO)
orco + or-1 + snmp-1 (+ BSA-)
5
24
14
25
11
11.25
8.129
15.33
18.42
0.0036
0.0172
0.0005
0.0001
0.0
0.248
2.275
0.5
0.0
0.0
1.241
4.0
1.0
2.381
6.769
4.0
9.342
3.5
5.0
non-transfected
or-1
orco
orco + or-1
orco + or-4
orco + or-1 (BAL 10-15 M)
orco + or-1 (DMSO)
orco + or-4 (DMSO)
orco + or-1 (BAL)
orco + or-4 (BAL)
orco (BAL)
or-1 (BAL)
non-transfected (BAL)
non-transfected (DMSO)
non-transfected (W7)
orco + or-1 (DMSO)
orco + or-1 (W7)
orco + or-1 (BAL)
DmelOrco + or-1 (BAL 10-12 M)
DmelOrco + or-1 (DMSO)
SF9 or-1 (BAL)
SF9 or-1 (DMSO)
SF9 or-4 (BAL)
SF9 or-4 (DMSO)
gα15 + or-1 (BAL)
gα15 + or-1 (DMSO)
gα15 + or-4 (BAL)
gα15 + or-4 (DMSO)
orco + or-1 + DmelSnmp-1 (BAL)
orco + or-1 + DmelSnmp-1 (DMSO)
orco + or-1 + snmp-1 (BAL ds. BAL-)
orco + or-1 + snmp-1 (BSA-)
30
13
13
36
36
17
12
4
18
17
8
36
14
18
17
35
32
10
11
24
5
5
3
4
4
3
3
3
3
3
5
10
5
10
6
6
9
5
21
17
17
12
7
5
40.20
17.10
60.59
60.59
10.84
3.650
19.14
39.13
17.62
60.59
16.52
24.15
45.19
71.84
33.45
3.829
9.270
39.08
0.0749
1.640
7.856
25.82
10.84
2.675
3.650
-
< 0.0001
0.0002
< 0.0001
< 0.0001
0.0044
0.1612
< 0.0001
< 0.0001
0.0001
< 0.0001
0.0003
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.1474
0.0097
< 0.0001
0.9632
0.4404
0.0197
< 0.0001
0.0044
0.2625
0.1612
-
0.0
0.0
0.0
0.0
0.0
2.571
0.243
0.0
0.0
0.0
1.618
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.446
0.0
0.403
0.883
0.0
0.0
0.0
0.0
1.594
2.571
0.0
0.243
2.174
0.0
0.0
0.0
0.0
0.0
0.0
3.846
3.154
0.0
0.0
0.0
6.226
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3.125
1.515
1.563
1.563
1.000
1.893
0.8197
1.697
1.000
1.000
0.9524
1.000
6.098
3.846
1.754
3.154
4.918
2.174
0.0
0.0
0.807
0.0
0.0
9.504
7.847
0.0
0.0
1.905
9.876
0.0
0.0
0.0
0.0
0.0
0.0
1.934
1.754
1.172
0.0
11.58
0.0
0.0
0.0
0.0
10.61
4.688
3.030
2.667
1.900
2.678
1.690
4.268
5.863
2.405
3.296
2.674
14.21
9.504
4.886
7.847
6.522
6.030
233
Appendix
97B
Fig.
97C
Fig.
97D
Fig.
98
Fig.
99B
Fig.
100B
Fig.
101C
Fig.
101D
Fig.
102B
Fig.
102C
Fig.
102D
Fig.
103B
Fig.
103C
Fig.
105B
Fig.
106B
Fig.
127B
orco + or-1 + DmelSnmp-1 (BAL ds. BSA-)
orco + or-1 + DmelSnmp-1 (BSA-)
orco + or-1 + snmp-1 (BAL 10-9 M)
orco + or-1 + snmp-1 (spontan.)
orco + or-1 + snmp-1 (BAL)
orco + or-1 + snmp-1 (C-15 ds. BSA-)
orco + or-1 + snmp-1 (BSA)
orco + or-1 + snmp-1 (C-15, + BSA-)
orco + or-1 + snmp-1 (spontan., + BSA-)
orco + or-4 + snmp-1 (C-15, + DMSO)
orco + or-4 + snmp-1 (spontan., + DMSO)
-orco PMA
-orco DMSO
+orco PMA
+orco DMSO
+orco FSK
+orco DMSO
13
35
4
4
24
9
25
10
13
5
11
4
5
6
14
6
11
9
25
11
11
5
5
8
8
3.244
14.44
11.25
4.189
15.33
25.18
7.571
18.42
8.129
18.42
1.543
15.33
31.15
31.15
2.176
22.96
0.1975
0.0007
0.0036
0.1231
0.0005
< 0.0001
0.0227
0.0001
0.0172
0.0001
0.4623
0.0005
< 0.0001
< 0.0001
0.3369
< 0.0001
1.704
2.597
0.850
0.250
0.248
1.125
0.500
1.399
2.626
2.132
0.0
1.087
0.0
1.482
2.275
0.0
0.0
1.480
0.500
0.0
0.0
0.0
0.0
0.0
0.0
4.082
4.211
1.500
7.500
1.241
2.041
1.000
3.159
4.000
4.545
2.381
4.348
2.174
2.806
4.000
3.571
2.381
6.579
1.000
0.0
0.0
0.9804
0.0
0.0
0.0
7.953
6.977
4.250
17.75
4.000
7.083
3.500
6.716
10.58
23.13
5.000
6.444
6.030
3.899
9.342
7.370
5.000
16.12
3.500
0.0
0.0
2.633
0.0
3.532
0.0
+orco 8-Br-cAMP
+orco DMSO
-orco 8-Br-cAMP
-orco DMSO
+orco 8-Br-cAMP
+orco DMSO
-orco 8-Br-cAMP
-orco DMSO
+orco 8-Br-cGMP 5 µM
-orco 8-Br-cGMP 5 µM
+orco 8-Br-cGMP
+orco DMSO
-orco 8-Br-cGMP
-orco DMSO
+orco 8-Br-cGMP
+orco DMSO
-orco 8-Br-cGMP
-orco DMSO
+orco VUAA1
+orco spontan.
-orco VUAA1
-orco spontan.
+orco VUAA1
+orco spontan.
-orco VUAA1
-orco spontan.
+orco DEET
+orco DMSO
17
49
13
43
9
9
12
12
20
5
10
3
25
66
13
43
18
18
13
13
71
65
23
30
43
43
20
20
8
8
12.74
81.90
18.39
61.80
2.008
1.949
25.06
22.49
25.06
25.89
107.6
32.54
61.80
29.37
12.33
32.54
36.07
32.18
29.58
22.24
35.42
28.03
29.83
17.36
35.87
3.840
-
0.0017
< 0.0001
0.0001
< 0.0001
0.3664
0.3773
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0021
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0002
< 0.0001
0.1466
-
0.769
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.000
0.0
0.0
0.0
1.000
0.0
0.0
0.0
0.0
0.0
2.353
0.0
0.0
0.0
1.613
1.613
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3.000
1.000
1.000
0.0
1.000
1.000
1.000
0.0
0.0
0.0
5.857
0.0
0.8065
0.0
5.812
10.52
0.0
0.0
3.512
4.223
1.232
0.0
3.452
0.0
0.5435
0.0
3.007
2.902
0.5435
0.0
14.00
3.000
2.000
1.250
6.333
3.000
2.205
1.000
4.610
0.0
+orco Ruthenium red
+orco DMSO
3
3
-
-
0.0
0.0
2.000
0.0
3.000
0.0
SF9 DMSO
SF9 FSK
10
10
0.728
1.339
0.6949
0.5121
0.605
0.811
0.9464
1.909
1.743
2.983
The K2 and p value computed by the D'Agostino-Pearson normality test as well as desriptive statistical parameters such as
th
th
median, 25 percentile, and 75 percentile are shown. Abbreviations: ds. = dissolved in, spontan. = spontaneous. For
further information see the respective figures.
234
Appendix
Tab. 31. Statistics of calcium imaging data (heterologous expression system) 2
Fig.
Data groups
Fig.
87C
ATP (low concentration)
ATP (high concentration)
DMSO
BSA, transfected cells
Fig.
88C
Fig.
89A
Fig.
89B
Fig.
90A
Fig.
90B
BSA(-) 10-6 M
BSA(-) 10-5 M
BSA(-) 10-4 M
BSA
DMSO
BSADMSO
non-transfected
orco + or-1
orco + or-1 + MsexSnmp-1
non-transfected
p value
(KW)
< 0.0001
-
Pairwise comparison
ATP (low conc.) vs. ATP (high conc.)
ATP (low conc.) vs. DMSO
ATP (low conc.) vs. DMSO
BSA, transfected cells vs.
-
-
-
BSA vs. DMSO
BSA vs. BSA(-)
BSA(-) vs. DMSO
DMSO vs. DMSO
non-transfected vs.
All other comparisons
MWT
MWT
MWT
MWT
Dunn's
< 0.0001
< 0.0001
0.6374
0.0002
< 0.05
Dunn's
> 0.05
non-transfected vs.
non-transfected vs.
orco + or-1 + snmp-1 vs.
orco + or-1 + snmp-1 vs.
Dunn's
< 0.05
Dunn's
< 0.05
Dunn's
> 0.05
MWT
0.2846
0.0081
0.0158
-
non-transfected (Ringer)
non-transfected (+ DMSO)
-
non-transfected (Ringer) vs.
non-transfected (DMSO)
MWT
0.3397
0.0224
orco + or-1 + snmp-1 (Ringer) vs.
orco + or-1 + snmp-1 (+DMSO)
orco + or-1 + snmp-1 (Ringer) vs.
orco + or-1 + snmp-1 (+BSA-)
orco + or-1 + snmp-1 (+DMSO) vs.
orco + or-1 + snmp-1 (+BSA-)
orco + or-4 + snmp-1 (+ DMSO) vs.
Dunn's
> 0.05
Dunn's
> 0.05
Dunn's
< 0.05
MWT
0.3137
non-transfected vs.
non-transfected vs.
or-1 vs.
or-1 vs.
orco vs.
orco + or-1 vs.
orco + or-1 vs.
orco + or-4 vs.
orco + or-4 vs.
orco + or-1 (BAL 10-15 M) vs.
orco + or-1 (BAL 10-6 M) vs. DMSO
Dunn's
< 0.001
Dunn's
< 0.05
Dunn's
< 0.001
Dunn's
< 0.01
Dunn's
< 0.01
Dunn's
< 0.001
Dunn's
< 0.05
Dunn's
< 0.001
Dunn's
< 0.05
Dunn's
Dunn's
> 0.05
< 0.01
Dunn's
< 0.01
Dunn's
< 0.05
Dunn's
< 0.001
orco + or-1 + snmp-1 (Ringer)
non-transfected
< 0.0001
or-1
orco
orco + or-1
orco + or-4
Fig.
92C
> 0.05
< 0.001
< 0.001
0.5338
-
Fig.
90F
Fig.
91
p value
0.9086
Fig.
90C
Fig.
90D
Fig.
90E
Pairwise
test
Dunn's
Dunn's
Dunn's
MWT
0.0006
235
Appendix
Fig.
92D
Fig.
92E
Fig.
95C
Fig.
96C
Fig.
97A
orco + or-1 (DMSO)
orco + or-4 (DMSO)
orco + or-1 (BAL)
orco + or-4 (BAL)
orco (BAL)
or-1 (BAL)
SF9 or-1 (BAL)
SF9 or-1 (DMSO)
SF9 or-4 (BAL)
SF9 or-4 (DMSO)
gα15 + or-1 (BAL)
gα15 + or-1 (DMSO)
gα15 + or-4 (BAL)
gα15 + or-4 (DMSO)
0.6490
-
Dunn's
-
> 0.05
-
0.3207
-
-
-
-
SF9 or-1 (BAL) vs. SF9 or-1 (DMSO)
SF9 or-1 (BAL) vs. SF9 or-4 (BAL)
SF9 or-4 (BAL) vs. SF9 or-4 (DMSO)
SF9 or-1 (DMSO) vs. SF9 or-4 (DMSO)
gα15 + or-1 (BAL) vs. gα15 + or-1 (DMSO)
gα15 + or-1 (BAL) vs. gα15 + or-4 (BAL)
gα15 + or-4 (BAL) vs. gα15 + or-4 (DMSO)
gα15 + or-1 (DMSO) vs. gα15 + or-4 (DMSO)
orco + or-1 + DmelSnmp-1 (BAL) vs.
orco + or-1 + DmelSnmp-1 (DMSO) vs.
orco + or-1 + snmp-1 (BAL ds. BSA-) vs.
orco + or-1 + snmp-1 (BAL ds. BSA-) vs.
vs.
orco + or-1 + snmp-1 (BSA-) vs.
-
MWT
MWT
MWT
MWT
MWT
MWT
MWT
MWT
MWT
0.3946
0.4916
0.1696
0.7821
0.6775
0.8570
0.5318
0.8422
0.5866
MWT
0.0082
MWT
0.2022
MWT
0.3512
MWT
0.4118
MWT
0.8740
MWT
0.7104
MWT
0.2520
-
-
orco + or-4 + snmp-1 (BAL 10-9 M) vs.
orco + or-1 + snmp-1 (BAL 10-9 M) vs.
orco + or-1 + snmp-1 (spontan.) vs.
orco + or-1 + snmp-1 (BAL) vs.
orco + or-1 + snmp-1 (spontan)
orco + or-1 + snmp-1 (spontan.) vs.
orco + or-1 + snmp-1 (C-15 ds. BSA-) vs.
orco + or-1 + snmp-1 (C-15, + BSA-) vs.
orco + or-4 + snmp-1 (C-15, + DMSO) vs.
orco + or-4 + snmp-1 (spontan., + DMSO)
MWT
0.2067
MWT
0.4583
MWT
0.9838
MWT
0.4380
MWT
0.5135
MWT
0.4278
MWT
0.1732
MWT
0.8016
MWT
0.2317
MWT
0.8787
MWT
0.0210
MWT
0.8099
MWT
0.2612
-orco PMA vs. -orco DMSO
+orco PMA vs. +orco DMSO
Wilcoxon
Wilcoxon
1.0000
0.2500
-
-
Fig.
97B
orco + or-1 + snmp-1 (BAL ds. BSA-)
-
(BAL ds. BSA-)
Fig.
97C
0.7147
-
Fig.
97D
-
Fig.
98B
orco + or-1 + snmp-1 (C-15 ds. BSA-)
-
Fig.
orco + or-4 + snmp-1 (spontan.,
+ BSA-)
orco + or-4 + snmp-1 (spontan.,
+ DMSO)
-orco PMA
-orco DMSO
-
236
Appendix
99B
Fig.
100B
Fig.
101C
Fig.
101D
Fig.
102B
Fig.
102C
Fig.
102D
Fig.
103B
Fig.
103C
Fig.
105B
Fig.
127B
+orco PMA
+orco DMSO
+orco FSK
+orco DMSO
-orco PMA vs. +orco PMA
-orco DMSO vs. +orco DMSO
+orco FSK vs. +orco DMSO
MWT
Wilcoxon
0.0741
1.0000
+orco 8-Br-cAMP vs. +orco DMSO
-orco 8-Br-cAMP vs. -orco DMSO
+orco 8-Br-cAMP vs. -orco 8-Br-cAMP
+orco DMSO vs. -orco DMSO
+orco 8-Br-cAMP vs. +orco DMSO
MWT
MWT
MWT
MWT
Wilcoxon
0.0003
0.3719
0.0044
0.2214
0.4375
-orco 8-Br-cAMP vs. -orco DMSO
Wilcoxon
0.5000
+orco 8-Br-cGMP 5 µM vs.
+orco 8-Br-cGMP 5 µM vs.
+orco 8-Br-cGMP vs. +orco DMSO
-orco 8-Br-cGMP vs. -orco DMSO
+orco 8-Br-cGMP vs. -orco 8-Br-cGMP
+orco DMSO vs. -orco DMSO
+orco 8-Br-cGMP vs. +orco DMSO
MWT
0.9084
MWT
0.4918
MWT
MWT
MWT
MWT
Wilcoxon
0.0340
0.3892
0.2444
0.4204
0.8457
-orco 8-Br-cGMP vs. -orco DMSO
Wilcoxon
0.2500
+orco VUAA1 vs. +orco spontan.
-orco VUAA1 vs. -orco spontan.
+orco VUAA1 vs. -orco VUAA1
+orco spontan. vs. -orco spontan.
+orco VUAA1 vs. +orco spontan.
MWT
MWT
MWT
MWT
Wilcoxon
0.0030
0.0546
0.0334
0.0184
0.0124
-orco VUAA1 vs. -orco spontan.
Wilcoxon
0.2227
-
+orco 8-Br-cAMP
+orco DMSO
-orco 8-Br-cAMP
-orco DMSO
+orco 8-Br-cAMP
+orco DMSO
-orco 8-Br-cAMP
-orco DMSO
+orco 8-Br-cGMP
+orco DMSO
-orco 8-Br-cGMP
-orco DMSO
+orco 8-Br-cGMP
+orco DMSO
-orco 8-Br-cGMP
-orco DMSO
+orco VUAA1
+orco spontan.
-orco VUAA1
-orco spontan.
+orco VUAA1
+orco spontan.
-orco VUAA1
-orco spontan.
+orco DEET
+orco DMSO
-
-
+orco DEET vs. +orco DMSO
Wilcoxon
0.2500
SF9 DMSO
SF9 FSK
-
SF9 DMSO vs. SF9 FSK
Wilcoxon
0.1289
-
-
-
-
-
-
P values of groupwise comparisons (Kruskal-Wallis test, KW) and/or (subsequent) pairwise comparisons are shown. Dunn's
post test was applied, when the Kruskal-Wallis test found significant differences between more than two data groups,
while Mann-Whitney test (MWT, for unpaired data) or Wilcoxon matched pairs test (for paired data) were applied for the
comparison of two data groups. Abbreviations: ds. = dissolved in, spontan. = spontaneous. For further information see the
respective figures.
237
Appendix
5.8 Calcium imaging experiments on primary M. sexta ORN
cell cultures
Fig. 128. The unspecific effects of MIA
on the fluorescence ratio in primary M.
sexta ORN cell cultures might be
partially
due
to
background
fluorescence changes. A. Normalized
calcium imaging data for 16 cells of a
primary M. sexta ORN culture. The data
represent
the
mean
percentage
deviation of the fluorescence ratio
(% Δ(F340/F380), mean ± SEM) from the
mean of the values before stimulation.
During application of the presumptive
ORCO antagonist MIA (30 µM, bar) the
cells showed increases of the
fluorescence ratio. After washout the
cells maintained a lower fluorescence
ratio. B, C. The normalized fluorescence
intensity, resulting from excitation at
340 nm (% Δ(F340), B) or 380 nm
(% Δ(F380), mean ± SEM, C), decreased
MIA-dependently. After washout the
mean F340 intensity maintained a slightly
lower level (B) and the mean F380
intensity a higher level (C). D, E. The
background fluorescence resulting from
excitation at 340 nm (% Δ(F340), D) did
not show MIA-dependent changes, while
the background fluorescence resulting
from excitation at 380 nm (% Δ(F380), E)
showed a mild MIA-dependent increase.
238
Appendix
Tab. 32. Statistics of calcium imaging data (primary ORN cell cultures) 1
Fig
Fig.
107C
Fig.
107D
Fig.
108C
Fig.
108D
Fig.
109B
Fig.
110B
Fig.
111B
Fig.
111C
Fig.
112D
Fig.
112E
Fig.
112F
Fig.
113B
Fig.
113C
< 0.0001
0.1404
0.0008
0.1168
0.1876
< 0.0001
< 0.0001
25th percentile
(%)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Median
(%)
0.0
0.0
0.0
0.0
3.721
3.125
0.0
0.0
0.0
0.0
0.0
75th percentile
(%)
0.0
1.829
2.941
1.786
13.94
7.692
0.0
4.762
0.0
2.851
2.985
-
-
0.0
0.0
0.0
0.0
1.500
3.000
6
6
-
-
0.0
0.0
0.0
3.571
0.8621
14.21
OLC15 (activation)
DMSO (activation)
6
6
-
-
0.0
0.0
0.0
0.0
4.554
6.452
OLC15 (inhibition)
DMSO (inhibition)
6
6
-
-
0.0
0.0
0.0
0.0
0.0
2.949
HMA 30µM (activation)
10
6
4.182
-
0.1235
-
7.084
0.0
11.86
25.32
31.98
49.22
HMA 30µM (inhibition)
HMA (activation)
DMSO (activation)
10
6
16
16
5.359
3.434
4.584
0.0686
0.1796
0.1011
2.954
3.261
6.617
0.0
5.253
6.513
14.84
1.282
11.95
11.82
39.97
7.555
HMA (inhibition)
DMSO (inhibition)
16
16
5.054
24.10
0.0799
< 0.0001
3.305
0.0
6.066
0.0
11.10
1.471
MIA (activation)
DMSO (activation)
16
16
19.56
4.584
< 0.0001
0.1011
0.4902
0.0
4.613
1.282
12.26
7.555
MIA (inhibition)
DMSO (inhibition)
16
16
25.60
24.10
< 0.0001
< 0.0001
0.7813
0.0
5.115
0.0
7.692
1.471
Data group
n
K2
p (norm.)
Ringer
Probenecid
Verapamil
Ringer DMSO
Probenecid DMSO
Verapamil DMSO
VUAA1 100µM
VUAA1 200µM
VUAA1 500µM
VUAA1
DMSO
9
8
3
10
8
3
7
11
12
28
28
25.84
3.926
14.28
4.295
3.347
20.68
20.17
OLC12
DMSO
4
4
VUAA4
DMSO
The K2 and p value computed by the D'Agostino-Pearson normality test as well as descriptive statistical parameters such
th
th
as median, 25 percentile, and 75 percentile are shown. For further information see the respective figures.
239
Appendix
Tab. 33. Statistics of calcium imaging data (primary ORN cell cultures) 2
Fig.
Fig.
107C
Fig.
107D
Fig.
108C
Fig.
108D
Fig.
109B
Fig.
110B
Fig.
111B
Fig.
111C
Fig.
112D
Fig.
112E
Fig.
112F
Fig.
113B
Fig.
113C
Data groups
p value
-
Pairwise
test
-
0.1952
-
-
-
0.0720
-
-
-
-
VUAA1 vs. DMSO
Wilcoxon
0.9515
OLC12
DMSO
-
OLC12 vs. DMSO
Wilcoxon
1.0000
VUAA4
DMSO
-
VUAA4 vs. DMSO
Wilcoxon
0.2500
OLC15 (activation)
DMSO (activation)
-
OLC15 vs. DMSO (activation)
Wilcoxon
1.0000
OLC15 (inhibition)
DMSO (inhibition)
-
OLC15 vs. DMSO (inhibition)
Wilcoxon
0.5000
-
HMA 30µM vs. HMA 60µM (activation)
MWT
0.9578
HMA (activation)
DMSO (activation)
-
HMA 30µM vs. HMA 60µM (inhibition)
MWT
0.8281
-
HMA vs. DMSO (activation)
Wilcoxon
0.0002
HMA (inhibition)
DMSO (inhibition)
-
HMA vs. DMSO (inhibition)
Wilcoxon
0.0017
MIA (activation)
DMSO (activation)
-
MIA vs. DMSO (activation)
Wilcoxon
0.0923
MIA (inhibition)
DMSO (inhibition)
-
MIA vs. DMSO (inhibition)
Wilcoxon
0.0010
Ringer
Probenecid
Verapamil
Ringer DMSO
Probenecid DMSO
Verapamil DMSO
VUAA1 100µM
VUAA1 200µM
VUAA1 500µM
VUAA1
DMSO
p value
(KW)
0.7053
Pairwise comparison
-
P values of groupwise comparisons (Kruskal-Wallis test, KW) or pairwise comparisons are shown. The Mann-Whitney test
(MWT) was applied for the comparison of two unpaired data groups and the Wilcoxon matched pairs test for the
comparison of two paired data groups. For further information see the respective figures.
240
Bibliography
6 Bibliography
1. Abdel-Latief M (2007) A family of chemoreceptors in Tribolium castaneum (Tenebrionidae:
Coleoptera). PLoS One 2 (12):e1319
2. Abrahamson EE, Moore RY (2001) Suprachiasmatic nucleus in the mouse: retinal innervation,
intrinsic organization and efferent projections. Brain Res 916 (1-2):172-191
3. Abuin L, Bargeton B, Ulbrich MH, Isacoff EY, Kellenberger S, Benton R (2011) Functional
architecture of olfactory ionotropic glutamate receptors. Neuron 69 (1):44-60
4. Agrawal T, Sadaf S, Hasan G (2013) A genetic RNAi screen for IP3/Ca2+ coupled GPCRs in Drosophila
identifies the PdfR as a regulator of insect flight. PLoS Genet 9 (10):e1003849
5. Ai M, Blais S, Park JY, Min S, Neubert TA, Suh GS (2013) Ionotropic glutamate receptors IR64a and
IR8a form a functional odorant receptor complex in vivo in Drosophila. J Neurosci 33
(26):10741-10749
6. Akten B, Jauch E, Genova GK, Kim EY, Edery I, Raabe T, Jackson FR (2003) A role for CK2 in the
Drosophila circadian oscillator. Nat Neurosci 6 (3):251-257
7. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2007) Molecular biology of the cell. 5th
edn. Taylor & Francis Ltd., Abingdon, UK
8. Alderton F, Sambi B, Tate R, Pyne NJ, Pyne S (2001) Assessment of agonism at G-protein coupled
receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293
cells. Br J Pharmacol 134 (1):6-9
9. Alix P, Grolleau F, Hue B (2002) Ca2+/calmodulin-dependent protein kinase regulates GABAactivated Cl- current in cockroach dorsal unpaired median neurons. J Neurophysiol 87
(6):2972-2982
10. Allada R, Kadener S, Nandakumar N, Rosbash M (2003) A recessive mutant of Drosophila Clock
reveals a role in circadian rhythm amplitude. EMBO J 22 (13):3367-3375
11. Allada R, White NE, So WV, Hall JC, Rosbash M (1998) A mutant Drosophila homolog of
mammalian Clock disrupts circadian rhythms and transcription of period and timeless. Cell 93
(5):791-804
12. An S, Irwin RP, Allen CN, Tsai C, Herzog ED (2011) Vasoactive intestinal polypeptide requires
parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a
predictable phase. J Neurophysiol 105 (5):2289-2296
13. An S, Tsai C, Ronecker J, Bayly A, Herzog ED (2012) Spatiotemporal distribution of vasoactive
intestinal polypeptide receptor 2 in mouse suprachiasmatic nucleus. J Comp Neurol 520
(12):2730-2741
14. Antolin S, Reisert J, Matthews HR (2010) Olfactory response termination involves Ca2+-ATPase in
vertebrate olfactory receptor neuron cilia. The Journal of General Physiology 135 (4):367-378
15. Armstrong D, Eckert R (1987) Voltage-activated calcium channels that must be phosphorylated to
respond to membrane depolarization. Proc Natl Acad Sci U S A 84 (8):2518-2522
16. Aschoff J (1979) Circadian Rhythms: Influences of Internal and External Factors on the Period
Measured in Constant Conditions1. Zeitschrift für Tierpsychologie 49 (3):225-249
17. Aschoff J (1984) Circadian timing. Ann N Y Acad Sci 423:442-468
18. Atkinson SE, Maywood ES, Chesham JE, Wozny C, Colwell CS, Hastings MH, Williams SR (2011)
Cyclic AMP signaling control of action potential firing rate and molecular circadian
pacemaking in the suprachiasmatic nucleus. J Biol Rhythms 26 (3):210-220
19. Aton SJ, Colwell CS, Harmar AJ, Waschek J, Herzog ED (2005) Vasoactive intestinal polypeptide
mediates circadian rhythmicity and synchrony in mammalian clock neurons. Nat Neurosci 8
(4):476-483
20. Bae K, Lee C, Sidote D, Chuang KY, Edery I (1998) Circadian regulation of a Drosophila homolog of
the mammalian Clock gene: PER and TIM function as positive regulators. Mol Cell Biol 18
(10):6142-6151
241
Bibliography
21. Baker TC, Cardé RT (1979) Endogenous and exogenous factors affecting periodicities of female
calling and male sex pheromone response in Grapholitha molesta (Busck). Journal of Insect
Physiology 25 (12):943-950
22. Baker TC, Haynes KF (1989) Field and laboratory electroantennographic measurements of
pheromone plume structure correlated with oriental fruit moth behaviour. Physiological
Entomology 14 (1):1-12
23. Barbara GS, Zube C, Rybak J, Gauthier M, Grunewald B (2005) Acetylcholine, GABA and glutamate
induce ionic currents in cultured antennal lobe neurons of the honeybee, Apis mellifera. J
Comp Physiol A Neuroethol Sens Neural Behav Physiol 191 (9):823-836
24. Barth M, Schultze M, Schuster CM, Strauss R (2010) Circadian plasticity in photoreceptor cells
controls visual coding efficiency in Drosophila melanogaster. PLoS One 5 (2):e9217
25. Bartsch M, Zorn-Kruppa M, Kuhl N, Genieser HG, Schwede F, Jastorff B (2003) Bioactivatable,
membrane-permeant analogs of cyclic nucleotides as biological tools for growth control of C6
glioma cells. Biol Chem 384 (9):1321-1326
26. Bau J, Justus KA, Carde RT (2002) Antennal resolution of pulsed pheromone plumes in three moth
species. J Insect Physiol 48 (4):433-442
27. Bau J, Justus KA, Loudon C, Carde RT (2005) Electroantennographic resolution of pulsed
pheromone plumes in two species of moths with bipectinate antennae. Chem Senses 30
(9):771-780
28. Baz E-S (2015) A molecular and cellular analysis of the circadian system of the cockroach
Rhyparobia (Leucophaea) maderae - The neuropeptide PDF and neurotransmitters involved
in input pathways to the circadian clock. PhD thesis, University of Kassel, Kassel
29. Baz E-S, Wei H, Grosshans J, Stengl M (2013) Calcium responses of circadian pacemaker neurons
of the cockroach Rhyparobia maderae to acetylcholine and histamine. J Comp Physiol A 199
(5):365-374
30. Beaver LM, Gvakharia BO, Vollintine TS, Hege DM, Stanewsky R, Giebultowicz JM (2002) Loss of
circadian clock function decreases reproductive fitness in males of Drosophila melanogaster.
Proc Natl Acad Sci U S A 99 (4):2134-2139
31. Becq F (1996) Ionic channel rundown in excised membrane patches. Biochimica et Biophysica
Acta (BBA) - Reviews on Biomembranes 1286 (1):53-63
32. Bell RA, Joachim FG (1976) Techniques for rearing laboratory colonies of tobacco hornworms and
pink bollworms. Ann entomol Coc Amer 69:365-373
33. Belles B, Malécot CO, Hescheler J, Trautwein W (1988) “Run-down” of the Ca2+ current during
long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular
calcium. Pflugers Arch 411 (4):353-360
34. Benito J, Zheng H, Hardin PE (2007) PDP1epsilon functions downstream of the circadian oscillator
to mediate behavioral rhythms. J Neurosci 27 (10):2539-2547
35. Benna C, Bonaccorsi S, Wulbeck C, Helfrich-Forster C, Gatti M, Kyriacou CP, Costa R, Sandrelli F
(2010) Drosophila timeless2 is required for chromosome stability and circadian
photoreception. Curr Biol 20 (4):346-352
36. Benna C, Scannapieco P, Piccin A, Sandrelli F, Zordan M, Rosato E, Kyriacou CP, Valle G, Costa R
(2000) A second timeless gene in Drosophila shares greater sequence similarity with
mammalian tim. Curr Biol 10 (14):R512-513
37. Benos DJ (1982) Amiloride: a molecular probe of sodium transport in tissues and cells. Am J
Physiol 242 (3):C131-145
38. Benquet P, Frere S, Pichon Y, Tiaho F (2000) Properties and development of calcium currents in
embryonic cockroach neurons. Neurosci Lett 294 (1):49-52
39. Benquet P, Le Guen J, Pichon Y, Tiaho F (2002) Differential involvement of Ca 2+ channels in
survival and neurite outgrowth of cultured embryonic cockroach brain neurons. J
Neurophysiol 88 (3):1475-1490
40. Benton R, Vannice KS, Vosshall LB (2007) An essential role for a CD36-related receptor in
pheromone detection in Drosophila. Nature 450 (7167):289-293
242
Bibliography
41. Benton R, Sachse S, Michnick SW, Vosshall LB (2006) Atypical membrane topology and
heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol 4 (2):e20
42. Benton R, Vannice KS, Gomez-Diaz C, Vosshall LB (2009) Variant ionotropic glutamate receptors
as chemosensory receptors in Drosophila. Cell 136 (1):149-162
43. Bezprozvanny I, Tsien RW (1995) Voltage-dependent blockade of diverse types of voltage-gated
Ca2+ channels expressed in Xenopus oocytes by the Ca2+ channel antagonist mibefradil (Ro
40-5967). Mol Pharmacol 48 (3):540-549
44. Blanchardon E, Grima B, Klarsfeld A, Chelot E, Hardin PE, Preat T, Rouyer F (2001) Defining the
role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and
eclosion by targeted genetic ablation and PERIOD protein overexpression. Eur J Neurosci 13
(5):871-888
45. Blau J, Young MW (1999) Cycling vrille expression is required for a functional Drosophila clock.
Cell 99 (6):661-671
46. Bobkov YV, Ache BW (2007) Block by amiloride derivatives of odor-evoked discharge in lobster
olfactory receptor neurons through action on a presumptive TRP channel. Chem Senses 32
(2):149-159
47. Boekhoff I, Raming K, Breer H (1990a) Pheromone-induced stimulation of inositol-trisphosphate
formation in insect antennae is mediated by G-proteins. Journal of Comparative Physiology B
160 (1):99-103
48. Boekhoff I, Strotmann J, Raming K, Tareilus E, Breer H (1990b) Odorant-sensitive phospholipase C
in insect antennae. Cell Signal 2 (1):49-56
49. Boekhoff I, Seifert E, Göggerle S, Lindemann M, Krüger BW, Breer H (1993) Pheromone-induced
second-messenger signaling in insect antennae. Insect Biochem Mol Biol 23 (7):757-762
50. Bohbot JD, Dickens JC (2010) Insect repellents: modulators of mosquito odorant receptor activity.
PLoS One 5 (8):e12138
51. Bohbot JD, Dickens JC (2012) Odorant receptor modulation: ternary paradigm for mode of action
of insect repellents. Neuropharmacology 62 (5-6):2086-2095
52. Bohbot JD, Fu L, Le TC, Chauhan KR, Cantrell CL, Dickens JC (2011) Multiple activities of insect
repellents on odorant receptors in mosquitoes. Med Vet Entomol 25 (4):436-444
53. Bonen A, Campbell SE, Benton CR, Chabowski A, Coort SL, Han XX, Koonen DP, Glatz JF, Luiken JJ
(2004) Regulation of fatty acid transport by fatty acid translocase/CD36. Proc Nutr Soc 63
(2):245-249
54. Boto T, Gomez-Diaz C, Alcorta E (2010) Expression analysis of the 3 G-protein subunits, Gα, Gβ,
and Gγ , in the olfactory receptor organs of adult Drosophila melanogaster. Chem Senses 35
(3):183-193
55. Bray S, Amrein H (2003) A putative Drosophila pheromone receptor expressed in male-specific
taste neurons is required for efficient courtship. Neuron 39 (6):1019-1029
56. Breer H, Boekhoff I, Tareilus E (1990) Rapid kinetics of second messenger formation in olfactory
transduction. Nature 345 (6270):65-68
57. Briggman JV, Graves JS, Spicer SS, Cragoe EJ, Jr. (1983) The intracellular localization of amiloride
in frog skin. Histochem J 15 (3):239-255
58. Brown TM, Colwell CS, Waschek JA, Piggins HD (2007) Disrupted neuronal activity rhythms in the
suprachiasmatic nuclei of vasoactive intestinal polypeptide-deficient mice. J Neurophysiol 97
(3):2553-2558
59. Brüggemann A, Pardo LA, Stuhmer W, Pongs O (1993) Ether-a-go-go encodes a voltage-gated
channel permeable to K+ and Ca2+ and modulated by cAMP. Nature 365 (6445):445-448
60. Brusius JS (2009) Elektrophysiologische Analyse der inneren Uhr der Schabe Leucophaea
maderae. Diploma thesis, Philipps-University, Marburg
61. Bugaj V, Alexeenko V, Zubov A, Glushankova L, Nikolaev A, Wang Z, Kaznacheyeva E,
Bezprozvanny I, Mozhayeva GN (2005) Functional properties of endogenous receptor- and
store-operated calcium influx channels in HEK293 cells. J Biol Chem 280 (17):16790-16797
243
Bibliography
62. Busza A, Emery-Le M, Rosbash M, Emery P (2004) Roles of the two Drosophila CRYPTOCHROME
structural domains in circadian photoreception. Science 304 (5676):1503-1506
63. Buzsáki G, Wang XJ (2012) Mechanisms of gamma oscillations. Annu Rev Neurosci 35:203-225
64. Byerly L, Hagiwara S (1982) Calcium currents in internally perfused nerve cell bodies of Limnea
stagnalis. J Physiol 322:503-528
65. Cagampang FR, Piggins HD, Sheward WJ, Harmar AJ, Coen CW (1998) Circadian changes in PACAP
type 1 (PAC1) receptor mRNA in the rat suprachiasmatic and supraoptic nuclei. Brain Res 813
(1):218-222
66. Cantrell AR, Smith RD, Goldin AL, Scheuer T, Catterall WA (1997) Dopaminergic modulation of
sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific
sites in the sodium channel  subunit. J Neurosci 17 (19):7330-7338
67. Cao G, Nitabach MN (2008) Circadian control of membrane excitability in Drosophila
melanogaster lateral ventral clock neurons. J Neurosci 28 (25):6493-6501
68. Cao G, Platisa J, Pieribone VA, Raccuglia D, Kunst M, Nitabach MN (2013) Genetically targeted
optical electrophysiology in intact neural circuits. Cell 154 (4):904-913
69. Castillo-Ruiz A, Paul MJ, Schwartz WJ (2012) In search of a temporal niche: social interactions.
Prog Brain Res 199:267-280
70. Catterall WA (1999) Molecular properties of brain sodium channels: an important target for
anticonvulsant drugs. Adv Neurol 79:441-456
71. Cayre M, Buckingham SD, Strambi A, Strambi C, Sattelle DB (1998) Adult insect mushroom body
neurons in primary culture: cell morphology and characterization of potassium channels. Cell
Tissue Res 291 (3):537-547
72. Ceriani MF, Darlington TK, Staknis D, Mas P, Petti AA, Weitz CJ, Kay SA (1999) Light-dependent
sequestration of TIMELESS by CRYPTOCHROME. Science 285 (5427):553-556
73. Chatterjee A, Hardin PE (2010) Time to taste: circadian clock function in the Drosophila gustatory
system. Fly (Austin) 4 (4):283-287
74. Chatterjee A, Roman G, Hardin PE (2009) Go contributes to olfactory reception in Drosophila
melanogaster. BMC Physiol 9:22
75. Chatterjee A, Tanoue S, Houl JH, Hardin PE (2010) Regulation of gustatory physiology and
appetitive behavior by the Drosophila circadian clock. Curr Biol 20 (4):300-309
76. Chen S, Luetje CW (2012) Identification of new agonists and antagonists of the insect odorant
receptor co-receptor subunit. PLoS One 7 (5):e36784
77. Chen S, Luetje CW (2013) Phenylthiophenecarboxamide antagonists of the olfactory receptor coreceptor subunit from a mosquito. PLoS One 8 (12):e84575
78. Cheng Y, Hardin PE (1998) Drosophila photoreceptors contain an autonomous circadian oscillator
that can function without period mRNA cycling. J Neurosci 18 (2):741-750
79. Chiang M, Strong JA, Asem EK (1993) Bovine serum albumin enhances calcium currents in chicken
granulosa cells. Mol Cell Endocrinol 94 (1):27-36
80. Choi C, Fortin JP, McCarthy E, Oksman L, Kopin AS, Nitabach MN (2009) Cellular dissection of
circadian peptide signals with genetically encoded membrane-tethered ligands. Curr Biol 19
(14):1167-1175
81. Choi C, Cao G, Tanenhaus AK, McCarthy EV, Jung M, Schleyer W, Shang Y, Rosbash M, Yin JC,
Nitabach MN (2012) Autoreceptor control of peptide/neurotransmitter corelease from PDF
neurons determines allocation of circadian activity in drosophila. Cell Rep 2 (2):332-344
82. Chouquet B, Debernard S, Bozzolan F, Solvar M, Maibeche-Coisne M, Lucas P (2009) A TRP
channel is expressed in Spodoptera littoralis antennae and is potentially involved in insect
olfactory transduction. Insect Mol Biol 18 (2):213-222
83. Chouquet B, Bozzolan F, Solvar M, Duportets L, Jacquin-Joly E, Lucas P, Debernard S (2008)
Molecular cloning and expression patterns of a putative olfactory diacylglycerol kinase from
the noctuid moth Spodoptera littoralis. Insect Mol Biol 17 (5):485-493
244
Bibliography
84. Chouquet B, Lucas P, Bozzolan F, Solvar M, Maibeche-Coisne M, Durand N, Debernard S (2010)
Molecular characterization of a phospholipase C β potentially involved in moth olfactory
transduction. Chem Senses 35 (5):363-373
85. Christensen T, Hildebrand J (1987) Male-specific, sex pheromone-selective projection neurons in
the antennal lobes of the moth Manduca sexta. Journal of Comparative Physiology A 160
(5):553-569
86. Christensen TA, Hildebrand JG (1997) Coincident stimulation with pheromone components
improves temporal pattern resolution in central olfactory neurons. J Neurophysiol 77 (2):775781
87. Christensen TA, Hildebrand JG, Tumlinson JH, Doolittle RE (1989) Sex Pheromone Blend of
Manduca sexta: Responses of Central Olfactory lnterneurons to Antennal Stimulation in Male
Moths. Archives of Insect Biochemistry and Physiology 10 (4):281-291
88. Chung BY, Kilman VL, Keath JR, Pitman JL, Allada R (2009) The GABA(A) receptor RDL acts in
peptidergic PDF neurons to promote sleep in Drosophila. Curr Biol 19 (5):386-390
89. Chyb S, Raghu P, Hardie RC (1999) Polyunsaturated fatty acids activate the Drosophila lightsensitive channels TRP and TRPL. Nature 397 (6716):255-259
90. Clyne PJ, Warr CG, Freeman MR, Lessing D, Kim J, Carlson JR (1999) A novel family of divergent
seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22
(2):327-338
91. Cohen LB, Salzberg BM, Davila HV, Ross WN, Landowne D, Waggoner AS, Wang CH (1974)
Changes in axon fluorescence during activity: Molecular probes of membrane potential. J
Membrain Biol 19 (1):1-36
92. Cole SR, Ashman LK, Ey PL (1987) Biotinylation: an alternative to radioiodination for the
identification of cell surface antigens in immunoprecipitates. Mol Immunol 24 (7):699-705
93. Coleman JA, Quazi F, Molday RS (2013) Mammalian P4-ATPases and ABC transporters and their
role in phospholipid transport. Biochimica et Biophysica Acta (BBA) - Molecular and Cell
Biology of Lipids 1831 (3):555-574
94. Collins B, Mazzoni EO, Stanewsky R, Blau J (2006) Drosophila CRYPTOCHROME is a circadian
transcriptional repressor. Curr Biol 16 (5):441-449
95. Colwell CS (2005) Bridging the gap: coupling single-cell oscillators in the suprachiasmatic nucleus.
Nat Neurosci 8 (1):10-12
96. Colwell CS, Page TL (1990) A circadian rhythm in neural activity can be recorded from the central
nervous system of the cockroach. J Comp Physiol A 166 (5):643-649
97. Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Liu X, Waschek JA (2003)
Disrupted circadian rhythms in VIP- and PHI-deficient mice. Am J Physiol Regul Integr Comp
Physiol 285 (5):R939-949
98. Cook DJ, Milligan JV (1972) Electrophysiology and histology of the medial neurosecretory cells in
adult male cockroaches, Periplaneta americana. Journal of Insect Physiology 18 (6):11971214
99. Corbel V, Stankiewicz M, Pennetier C, Fournier D, Stojan J, Girard E, Dimitrov M, Molgo J,
Hougard JM, Lapied B (2009) Evidence for inhibition of cholinesterases in insect and
mammalian nervous systems by the insect repellent DEET. BMC Biol 7:47
100. Corey DP, Hudspeth AJ (1979) Response latency of vertebrate hair cells. Biophys J 26 (3):499-506
101. Corey DP, Hudspeth AJ (1983) Kinetics of the receptor current in bullfrog saccular hair cells. J
Neurosci 3 (5):962-976
102. Corey EA, Bobkov Y, Ukhanov K, Ache BW (2013) Ionotropic crustacean olfactory receptors. PLoS
One 8 (4):e60551
103. Cornwell PB (1968) The cockroach. Volume 1., A laboratory insect and an industrial pest.
Hutchinson,
104. Couto A, Alenius M, Dickson BJ (2005) Molecular, anatomical, and functional organization of the
Drosophila olfactory system. Curr Biol 15 (17):1535-1547
245
Bibliography
105. Couvineau A, Laburthe M (2012) VPAC receptors: structure, molecular pharmacology and
interaction with accessory proteins. Br J Pharmacol 166 (1):42-50
106. Craven KB, Zagotta WN (2006) CNG and HCN channels: two peas, one pod. Annu Rev Physiol
68:375-401
107. Croset V, Rytz R, Cummins SF, Budd A, Brawand D, Kaessmann H, Gibson TJ, Benton R (2010)
Ancient protostome origin of chemosensory ionotropic glutamate receptors and the
evolution of insect taste and olfaction. PLoS Genet 6 (8):e1001064
108. Cully DF, Paress PS, Liu KK, Schaeffer JM, Arena JP (1996) Identification of a Drosophila
melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent
avermectin. J Biol Chem 271 (33):20187-20191
109. Curtin KD, Huang ZJ, Rosbash M (1995) Temporally regulated nuclear entry of the Drosophila
period protein contributes to the circadian clock. Neuron 14 (2):365-372
110. Cutler DJ, Haraura M, Reed HE, Shen S, Sheward WJ, Morrison CF, Marston HM, Harmar AJ,
Piggins HD (2003) The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular
rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro. Eur J Neurosci
17 (2):197-204
111. Cyran SA, Buchsbaum AM, Reddy KL, Lin MC, Glossop NR, Hardin PE, Young MW, Storti RV, Blau
J (2003) vrille, Pdp1, and dClock form a second feedback loop in the Drosophila circadian
clock. Cell 112 (3):329-341
112. Daaka Y, Luttrell LM, Lefkowitz RJ (1997) Switching of the coupling of the 2-adrenergic receptor
to different G proteins by protein kinase A. Nature 390 (6655):88-91
113. Daly KC, Kalwar F, Hatfield M, Staudacher E, Bradley SP (2013) Odor detection in Manduca sexta
is optimized when odor stimuli are pulsed at a frequency matching the wing beat during
flight. PLoS One 8 (11):e81863
114. Dardente H, Menet JS, Challet E, Tournier BB, Pevet P, Masson-Pevet M (2004) Daily and
circadian expression of neuropeptides in the suprachiasmatic nuclei of nocturnal and diurnal
rodents. Brain Res Mol Brain Res 124 (2):143-151
115. Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi
JS, Kay SA (1998) Closing the circadian loop: CLOCK-induced transcription of its own
inhibitors per and tim. Science 280 (5369):1599-1603
116. David JA, Pitman RM (1995) Muscarinic agonists modulate calcium-dependent outward currents
in an identified insect motoneurone. Brain Res 669 (1):153-156
117. Davis EE, Sokolove PG (1976) Lactic acid-sensitive receptors on the antennae of the
mosquito,<i>Aedes aegypti</i&gt. Journal of Comparative Physiology A:
Neuroethology, Sensory, Neural, and Behavioral Physiology 105 (1):43-54
118. de Bruyne M, Clyne PJ, Carlson JR (1999) Odor coding in a model olfactory organ: the Drosophila
maxillary palp. J Neurosci 19 (11):4520-4532
119. de Bruyne M, Foster K, Carlson JR (2001) Odor coding in the Drosophila antenna. Neuron 30
(2):537-552
120. de Mairan JJdO (1729) Observation botanique. Histoire de l’Académie Royale des Sciences
Paris:35-36
121. DeGennaro M, McBride CS, Seeholzer L, Nakagawa T, Dennis EJ, Goldman C, Jasinskiene N,
James AA, Vosshall LB (2013) orco mutant mosquitoes lose strong preference for humans
and are not repelled by volatile DEET. Nature
122. Delgado R, Latorre R, Labarca P (1995) Selectivity and gating properties of a cAMP-modulated,
K+-selective channel from Drosophila larval muscle. FEBS Lett 370 (1-2):113-117
123. Deng Y, Zhang W, Farhat K, Oberland S, Gisselmann G, Neuhaus EM (2011) The stimulatory Gαs
protein is involved in olfactory signal transduction in Drosophila. PLoS One 6 (4):e18605
124. Depetris-Chauvin A, Berni J, Aranovich EJ, Muraro NI, Beckwith EJ, Ceriani MF (2011) Adultspecific electrical silencing of pacemaker neurons uncouples molecular clock from circadian
outputs. Curr Biol 21 (21):1783-1793
246
Bibliography
125. Dibner C, Schibler U, Albrecht U (2010) The mammalian circadian timing system: organization
and coordination of central and peripheral clocks. Annu Rev Physiol 72:517-549
126. Dircksen H, Zahnow C, Gaus G, Keller R, Rao KR, Riehm J (1987) The ultrastructure of nerve
endings containing pigment-dispersing hormone (PDH) in crustacean sinus glands:
Identification by an antiserum against a synthetic PDH. Cell and Tissue Research 250 (2):377387
127. Ditzen M, Pellegrino M, Vosshall LB (2008) Insect odorant receptors are molecular targets of the
insect repellent DEET. Science 319 (5871):1838-1842
128. Dobritsa AA, van der Goes van Naters W, Warr CG, Steinbrecht RA, Carlson JR (2003) Integrating
the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37 (5):827841
129. Dogan EB, Ayres JW, Rossignol PA (1999) Behavioural mode of action of deet: inhibition of lactic
acid attraction. Med Vet Entomol 13 (1):97-100
130. Dolzer J (2002) Mechanisms of modulation and adaptation in pheromone-sensitive trichoid
sensilla of the hawkmoth Manduca sexta. PhD thesis, Philipps University, Marburg
131. Dolzer J, Fischer K, Stengl M (2003) Adaptation in pheromone-sensitive trichoid sensilla of the
hawkmoth Manduca sexta. J Exp Biol 206 (Pt 9):1575-1588
132. Dolzer J, Krannich S, Stengl M (2008) Pharmacological investigation of protein kinase C- and
cGMP-dependent ion channels in cultured olfactory receptor neurons of the hawkmoth
Manduca sexta. Chem Senses 33 (9):803-813
133. Dolzer J, Krannich S, Fischer K, Stengl M (2001) Oscillations of the transepithelial potential of
moth olfactory sensilla are influenced by octopamine and serotonin. J Exp Biol 204 (Pt
16):2781-2794
134. Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis 34 (1-2):115
135. Dushay MS, Rosbash M, Hall JC (1989) The disconnected visual system mutations in Drosophila
melanogaster drastically disrupt circadian rhythms. J Biol Rhythms 4 (1):1-27
136. Duvall LB, Taghert PH (2012) The circadian neuropeptide PDF signals preferentially through a
specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila. PLoS Biol 10
(6):e1001337
137. Duvall LB, Taghert PH (2013) E and M circadian pacemaker neurons use different PDF receptor
signalosome components in Drosophila. J Biol Rhythms 28 (4):239-248
138. Ebbs ML, Amrein H (2007) Taste and pheromone perception in the fruit fly Drosophila
melanogaster. Pflugers Arch 454 (5):735-747
139. Edery I, Zwiebel LJ, Dembinska ME, Rosbash M (1994) Temporal phosphorylation of the
Drosophila period protein. Proc Natl Acad Sci U S A 91 (6):2260-2264
140. Egan TM, Dagan D, Kupper J, Levitan IB (1992) Properties and rundown of sodium-activated
potassium channels in rat olfactory bulb neurons. J Neurosci 12 (5):1964-1976
141. Ehnbom K (1948) Studies on the central and sympathetic nervous system and some sense
organs in the head of neuropteroid insects. C.W.K. Gleerup,
142. Ehrlich I, Elmslie KS (1995) Neurotransmitters acting via different G proteins inhibit N-type
calcium current by an identical mechanism in rat sympathetic neurons. J Neurophysiol 74
(6):2251-2257
143. Eide PE, Caldwell JM, Marks EP (1975) Establishment of two cell lines from embryonic tissue of
the tobacco hornworm, Manduca sexta (L). In Vitro 11 (6):395-399
144. Eiras S, Camina JP, Diaz-Rodriguez E, Gualillo O, Casanueva FF (2004) Leptin inhibits
lysophosphatidic acid-induced intracellular calcium rise by a protein kinase C-dependent
mechanism. J Cell Physiol 201 (2):214-226
145. Elmore T, Ignell R, Carlson JR, Smith DP (2003) Targeted mutation of a Drosophila odor receptor
defines receptor requirement in a novel class of sensillum. J Neurosci 23 (30):9906-9912
146. Emery IF, Noveral JM, Jamison CF, Siwicki KK (1997) Rhythms of Drosophila period gene
expression in culture. Proc Natl Acad Sci U S A 94 (8):4092-4096
247
Bibliography
147. Emery P, Stanewsky R, Hall JC, Rosbash M (2000a) A unique circadian-rhythm photoreceptor.
Nature 404 (6777):456-457
148. Emery P, So WV, Kaneko M, Hall JC, Rosbash M (1998) CRY, a Drosophila clock and lightregulated cryptochrome, is a major contributor to circadian rhythm resetting and
photosensitivity. Cell 95 (5):669-679
149. Emery P, Stanewsky R, Helfrich-Forster C, Emery-Le M, Hall JC, Rosbash M (2000b) Drosophila
CRY is a deep brain circadian photoreceptor. Neuron 26 (2):493-504
150. Enserink JM, Christensen AE, de Rooij J, van Triest M, Schwede F, Genieser HG, Doskeland SO,
Blank JL, Bos JL (2002) A novel Epac-specific cAMP analogue demonstrates independent
regulation of Rap1 and ERK. Nat Cell Biol 4 (11):901-906
151. Ewer J, Frisch B, Hamblen-Coyle MJ, Rosbash M, Hall JC (1992) Expression of the period clock
gene within different cell types in the brain of Drosophila adults and mosaic analysis of these
cells' influence on circadian behavioral rhythms. J Neurosci 12 (9):3321-3349
152. Fabricius JC (1781) Species insectorum, exhibentes eorum differentias specificas, synonyma
auctorum, loca natalia, metamorphosin, adjectis observationibus, descriptionibus, vol t.1
(1781). impensis C. E. Bohnii, Hamburgi et Kilonii
153. Fang Y, Sathyanarayanan S, Sehgal A (2007) Post-translational regulation of the Drosophila
circadian clock requires protein phosphatase 1 (PP1). Genes Dev 21 (12):1506-1518
154. Fenwick EM, Marty A, Neher E (1982) Sodium and calcium channels in bovine chromaffin cells. J
Physiol 331:599-635
155. Fernandez MP, Berni J, Ceriani MF (2008) Circadian remodeling of neuronal circuits involved in
rhythmic behavior. PLoS Biol 6 (3):e69
156. Fernandez MP, Chu J, Villella A, Atkinson N, Kay SA, Ceriani MF (2007) Impaired clock output by
altered connectivity in the circadian network. Proc Natl Acad Sci U S A 104 (13):5650-5655
157. Ferveur JF (2005) Cuticular hydrocarbons: their evolution and roles in Drosophila pheromonal
communication. Behav Genet 35 (3):279-295
158. Flecke C, Stengl M (2009) Octopamine and tyramine modulate pheromone-sensitive olfactory
sensilla of the hawkmoth Manduca sexta in a time-dependent manner. J Comp Physiol A
Neuroethol Sens Neural Behav Physiol 195 (6):529-545
159. Flecke C, Nolte A, Stengl M (2010) Perfusion with cAMP analogue affects pheromone-sensitive
trichoid sensilla of the hawkmoth Manduca sexta in a time-dependent manner. J Exp Biol 213
(Pt 5):842-852
160. Flecke C, Dolzer J, Krannich S, Stengl M (2006) Perfusion with cGMP analogue adapts the action
potential response of pheromone-sensitive sensilla trichoidea of the hawkmoth Manduca
sexta in a daytime-dependent manner. J Exp Biol 209 (Pt 19):3898-3912
161. Fleissner G, Frisch B (1993) A new type of putative non-visual photoreceptors in the optic lobe
of beetles. Cell Tissue Res 273 (3):435-445
162. Fogle KJ, Parson KG, Dahm NA, Holmes TC (2011) CRYPTOCHROME is a blue-light sensor that
regulates neuronal firing rate. Science 331 (6023):1409-1413
163. Folmer DE, Elferink RPJO, Paulusma CC (2009) P4 ATPases - Lipid flippases and their role in
disease. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1791
(7):628-635
164. Forstner M, Breer H, Krieger J (2009) A receptor and binding protein interplay in the detection of
a distinct pheromone component in the silkmoth Antheraea polyphemus. Int J Biol Sci 5
(7):745-757
165. Forstner M, Gohl T, Breer H, Krieger J (2006) Candidate pheromone binding proteins of the
silkmoth Bombyx mori. Invert Neurosci 6 (4):177-187
166. Forstner M, Gohl T, Gondesen I, Raming K, Breer H, Krieger J (2008) Differential expression of
SNMP-1 and SNMP-2 proteins in pheromone-sensitive hairs of moths. Chem Senses 33
(3):291-299
167. French AS (1989) Two components of rapid sensory adaptation in a cockroach mechanoreceptor
neuron. J Neurophysiol 62 (3):768-777
248
Bibliography
168. Frings S, Lynch JW, Lindemann B (1992) Properties of cyclic nucleotide-gated channels mediating
olfactory transduction. Activation, selectivity, and blockage. J Gen Physiol 100 (1):45-67
169. Frisch B, Hardin PE, Hamblen-Coyle MJ, Rosbash M, Hall JC (1994) A promoterless period gene
mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the
Drosophila nervous system. Neuron 12 (3):555-570
170. Fujii S, Amrein H (2010) Ventral lateral and DN1 clock neurons mediate distinct properties of
male sex drive rhythm in Drosophila. Proc Natl Acad Sci U S A 107 (23):10590-10595
171. Funk NW (2005) Untersuchungen zur inneren Uhr der Schabe Leucophaea maderae Elektrophysiologische und molekularbiologische Analyse der circadianen Kopplung. Diploma
thesis, Philipps-University, Marburg, Germany
172. Gamper N, Reznikov V, Yamada Y, Yang J, Shapiro MS (2004) Phosphatidylinositol 4,5bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+
channels. J Neurosci 24 (48):10980-10992
173. Garcia RL, Schilling WP (1997) Differential expression of mammalian TRP homologues across
tissues and cell lines. Biochem Biophys Res Commun 239 (1):279-283
174. Ge Y, Elghetany MT (2005) CD36: a multiligand molecule. Lab Hematol 11 (1):31-37
175. Gekakis N, Saez L, Delahaye-Brown AM, Myers MP, Sehgal A, Young MW, Weitz CJ (1995)
Isolation of timeless by PER protein interaction: defective interaction between timeless
protein and long-period mutant PERL. Science 270 (5237):811-815
176. German PF, van der Poel S, Carraher C, Kralicek AV, Newcomb RD (2013) Insights into subunit
interactions within the insect olfactory receptor complex using FRET. Insect Biochem Mol Biol
43 (2):138-145
177. Getahun MN (2013) Response dynamics in Drosophila olfaction. PhD thesis, Friedrich Schiller
University, Jena
178. Getahun MN, Wicher D, Hansson BS, Olsson SB (2012) Temporal response dynamics of
Drosophila olfactory sensory neurons depends on receptor type and response polarity. Front
Cell Neurosci 6:54
179. Getahun MN, Olsson SB, Lavista-Llanos S, Hansson BS, Wicher D (2013) Insect odorant response
sensitivity is tuned by metabotropically autoregulated olfactory receptors. PLoS One 8
(3):e58889
180. Giebultowicz JM (2000) Molecular mechanism and cellular distribution of insect circadian clocks.
Annu Rev Entomol 45:769-793
181. Giebultowicz JM, Hege DM (1997) Circadian clock in Malpighian tubules. Nature 386 (6626):664
182. Giebultowicz JM, Riemann JG, Raina AK, Ridgway RL (1989) Circadian system controlling release
of sperm in the insect testes. Science 245 (4922):1098-1100
183. Giebultowicz JM, Stanewsky R, Hall JC, Hege DM (2000) Transplanted Drosophila excretory
tubules maintain circadian clock cycling out of phase with the host. Curr Biol 10 (2):107-110
184. Giniger E, Varnum SM, Ptashne M (1985) Specific DNA binding of GAL4, a positive regulatory
protein of yeast. Cell 40 (4):767-774
185. Glaser FT, Stanewsky R (2005) Temperature synchronization of the Drosophila circadian clock.
Curr Biol 15 (15):1352-1363
186. Gloerich M, Bos JL (2010) Epac: defining a new mechanism for cAMP action. Annu Rev
Pharmacol Toxicol 50:355-375
187. Glossop NR, Hardin PE (2002) Central and peripheral circadian oscillator mechanisms in flies and
mammals. J Cell Sci 115 (Pt 17):3369-3377
188. Glossop NR, Houl JH, Zheng H, Ng FS, Dudek SM, Hardin PE (2003) VRILLE feeds back to control
circadian transcription of Clock in the Drosophila circadian oscillator. Neuron 37 (2):249-261
189. Gocht D, Wagner S, Heinrich R (2009) Recognition, presence, and survival of locust central
nervous glia in situ and in vitro. Microsc Res Tech 72 (5):385-397
190. Golden SS, Ishiura M, Johnson CH, Kondo T (1997) Cyanobacterial circadian rhythms. Annu Rev
Plant Physiol Plant Mol Biol 48:327-354
249
Bibliography
191. Goldman AL, Van der Goes van Naters W, Lessing D, Warr CG, Carlson JR (2005) Coexpression of
two functional odor receptors in one neuron. Neuron 45 (5):661-666
192. Gomez-Diaz C, Martin F, Alcorta E (2004) The cAMP transduction cascade mediates olfactory
reception in Drosophila melanogaster. Behav Genet 34 (4):395-406
193. Gomez-Diaz C, Martin F, Alcorta E (2006) The Inositol 1,4,5-triphosphate kinase1 gene affects
olfactory reception in Drosophila melanogaster. Behav Genet 36 (2):309-321
194. Gorostiza EA, Ceriani MF (2013) Retrograde bone morphogenetic protein signaling shapes a key
circadian pacemaker circuit. J Neurosci 33 (2):687-696
195. Gorostiza EA, Depetris-Chauvin A, Frenkel L, Pírez N, Ceriani María F (2014) Circadian pacemaker
neurons change synaptic contacts across the day. Current Biology 24 (18):2161-2167
196. Granados-Fuentes D, Prolo LM, Abraham U, Herzog ED (2004) The suprachiasmatic nucleus
entrains, but does not sustain, circadian rhythmicity in the olfactory bulb. J Neurosci 24
(3):615-619
197. Grant PR (2005) The priming of periodical cicada life cycles. Trends Ecol Evol 20 (4):169-174
198. Grima B, Chelot E, Xia R, Rouyer F (2004) Morning and evening peaks of activity rely on different
clock neurons of the Drosophila brain. Nature 431 (7010):869-873
199. Groh KC, Vogel H, Stensmyr MC, Grosse-Wilde E, Hansson BS (2013) The hermit crab's noseantennal transcriptomics. Front Neurosci 7:266
200. Grolleau F, Lapied B (1994) Transient Na+-activated K+ current in beating pacemaker-isolated
adult insect neurosecretory cells (DUM neurones). Neurosci Lett 167 (1-2):46-50
201. Grolleau F, Lapied B (1995) Separation and identification of multiple potassium currents
regulating the pacemaker activity of insect neurosecretory cells (DUM neurons). J
202. Groschner K, Hingel S, Lintschinger B, Balzer M, Romanin C, Zhu X, Schreibmayer W (1998) Trp
proteins form store-operated cation channels in human vascular endothelial cells. FEBS Lett
437 (1-2):101-106
203. Grosse-Wilde E, Svatoš A, Krieger J (2006) A pheromone-binding protein mediates the
bombykol-induced activation of a pheromone receptor in vitro. Chem Senses 31 (6):547-555
204. Grosse-Wilde E, Gohl T, Bouche E, Breer H, Krieger J (2007) Candidate pheromone receptors
provide the basis for the response of distinct antennal neurons to pheromonal compounds.
Eur J Neurosci 25 (8):2364-2373
205. Grosse-Wilde E, Stieber R, Forstner M, Krieger J, Wicher D, Hansson BS (2010) Sex-specific
odorant receptors of the tobacco hornworm Manduca sexta. Front Cell Neurosci 4:22
206. Grosse-Wilde E, Kuebler LS, Bucks S, Vogel H, Wicher D, Hansson BS (2011) Antennal
transcriptome of Manduca sexta. Proc Natl Acad Sci U S A 108 (18):7449-7454
207. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly
improved fluorescence properties. J Biol Chem 260 (6):3440-3450
208. Guo S, Kim J (2010) Dissecting the molecular mechanism of Drosophila odorant receptors
through activity modeling and comparative analysis. Proteins 78 (2):381-399
209. Gvakharia BO, Kilgore JA, Bebas P, Giebultowicz JM (2000) Temporal and spatial expression of
the period gene in the reproductive system of the codling moth. J Biol Rhythms 15 (1):4-12
210. Ha TS, Smith DP (2006) A pheromone receptor mediates 11-cis-vaccenyl acetate-induced
responses in Drosophila. J Neurosci 26 (34):8727-8733
211. Ha TS, Xia R, Zhang H, Jin X, Smith DP (2014) Lipid flippase modulates olfactory receptor
expression and odorant sensitivity in Drosophila. Proc Natl Acad Sci U S A 111 (21):7831-7836
212. Hagberg M (1986) Ultrastructure and central projections of extraocular photoreceptors in
caddiesflies (Insecta: Trichoptera). Cell and Tissue Research 245 (3):643-648
213. Hallberg E, Hansson BS (1999) Arthropod sensilla: morphology and phylogenetic considerations.
Microsc Res Tech 47 (6):428-439
214. Hallem EA, Ho MG, Carlson JR (2004a) The molecular basis of odor coding in the Drosophila
antenna. Cell 117 (7):965-979
250
Bibliography
215. Hallem EA, Nicole Fox A, Zwiebel LJ, Carlson JR (2004b) Olfaction: mosquito receptor for humansweat odorant. Nature 427 (6971):212-213
216. Halling DB, Aracena-Parks P, Hamilton SL (2006) Regulation of voltage-gated Ca2+ channels by
calmodulin. Sci STKE 2006 (318):er1
217. Hamasaka Y, Mohrherr CJ, Predel R, Wegener C (2005) Chronobiological analysis and mass
spectrometric characterization of pigment-dispersing factor in the cockroach Leucophaea
maderae. J Insect Sci 5:43
218. Hamasaka Y, Rieger D, Parmentier ML, Grau Y, Helfrich-Förster C, Nässel DR (2007) Glutamate
and its metabotropic receptor in Drosophila clock neuron circuits. J Comp Neurol 505 (1):3245
219. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques
for high-resolution current recording from cells and cell-free membrane patches. Pflugers
Arch 391 (2):85-100
220. Hamilton EE, Kay SA (2008) SnapShot: circadian clock proteins. Cell 135 (2):368-368 e361
221. Hao H, Allen DL, Hardin PE (1997) A circadian enhancer mediates PER-dependent mRNA cycling
in Drosophila melanogaster. Mol Cell Biol 17 (7):3687-3693
222. Hao LY, Kameyama A, Kameyama M (1999) A cytoplasmic factor, calpastatin and ATP together
reverse run-down of Ca2+ channel activity in guinea-pig heart. J Physiol 514 ( Pt 3):687-699
223. Hardie RC, Raghu P (2001) Visual transduction in Drosophila. Nature 413 (6852):186-193
224. Hardin PE (1994) Analysis of period mRNA cycling in Drosophila head and body tissues indicates
that body oscillators behave differently from head oscillators. Mol Cell Biol 14 (11):72117218
225. Hardin PE (2011) Molecular genetic analysis of circadian timekeeping in Drosophila. Adv Genet
74:141-173
226. Hardin PE, Hall JC, Rosbash M (1990) Feedback of the Drosophila period gene product on
circadian cycling of its messenger RNA levels. Nature 343 (6258):536-540
227. Harini K, Sowdhamini R (2012) Molecular modelling of oligomeric states of DmOR83b, an
olfactory receptor in D. melanogaster. Bioinform Biol Insights 6:33-47
228. Harmar AJ (2003) An essential role for peptidergic signalling in the control of circadian rhythms
in the suprachiasmatic nuclei. J Neuroendocrinol 15 (4):335-338
229. Harmar AJ, Fahrenkrug J, Gozes I, Laburthe M, May V, Pisegna JR, Vaudry D, Vaudry H, Waschek
JA, Said SI (2012) Pharmacology and functions of receptors for vasoactive intestinal peptide
and pituitary adenylate cyclase-activating polypeptide: IUPHAR review 1. Br J Pharmacol 166
(1):4-17
230. Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P,
Said SI, Sreedharan SP, Wank SA, Waschek JA (1998) International Union of Pharmacology.
XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate
cyclase-activating polypeptide. Pharmacol Rev 50 (2):265-270
231. Harmar AJ, Marston HM, Shen S, Spratt C, West KM, Sheward WJ, Morrison CF, Dorin JR, Piggins
HD, Reubi JC, Kelly JS, Maywood ES, Hastings MH (2002) The VPAC2 receptor is essential for
circadian function in the mouse suprachiasmatic nuclei. Cell 109 (4):497-508
232. Harrisingh MC, Wu Y, Lnenicka GA, Nitabach MN (2007) Intracellular Ca2+ regulates free-running
circadian clock oscillation in vivo. J Neurosci 27 (46):12489-12499
233. Haug T, Storm JF (2000) Protein kinase A mediates the modulation of the slow Ca 2+-dependent
K+ current, IsAHP, by the neuropeptides CRF, VIP, and CGRP in hippocampal pyramidal
neurons. J Neurophysiol 83 (4):2071-2079
234. Hayashi K, Endoh T, Suzuki T (1999) VIP inhibits high voltage-gated calcium channel currents of
hamster submandibular ganglion neurons. Bull Tokyo Dent Coll 40 (2):93-97
235. Hayashi K, Endoh T, Shibukawa Y, Yamamoto T, Suzuki T (2002) VIP and PACAP inhibit L-, N- and
P/Q-type Ca2+ channels of parasympathetic neurons in a voltage independent manner. Bull
Tokyo Dent Coll 43 (1):31-39
251
Bibliography
236. Hege DM, Stanewsky R, Hall JC, Giebultowicz JM (1997) Rhythmic expression of a PER-reporter
in the Malpighian tubules of decapitated Drosophila: evidence for a brain-independent
circadian clock. J Biol Rhythms 12 (4):300-308
237. Helfrich-Förster C (1995) The period clock gene is expressed in central nervous system neurons
which also produce a neuropeptide that reveals the projections of circadian pacemaker cells
within the brain of Drosophila melanogaster. Proc Natl Acad Sci U S A 92 (2):612-616
238. Helfrich-Förster C (1998) Robust circadian rhythmicity of Drosophila melanogaster requires the
presence of lateral neurons: a brain-behavioral study of disconnected mutants. J Comp
Physiol A 182 (4):435-453
239. Helfrich-Förster C (2000) Differential control of morning and evening components in the activity
rhythm of Drosophila melanogaster - Sex-specific differences suggest a different quality of
activity. J Biol Rhythms 15 (2):135-154
240. Helfrich-Förster C (2014) From neurogenetic studies in the fly brain to a concept in circadian
biology. J Neurogenet 0 (0):1-19
241. Helfrich-Förster C, Homberg U (1993) Pigment-dispersing hormone-immunoreactive neurons in
the nervous system of wild-type Drosophila melanogaster and of several mutants with
altered circadian rhythmicity. J Comp Neurol 337 (2):177-190
242. Helfrich-Förster C, Winter C, Hofbauer A, Hall JC, Stanewsky R (2001) The circadian clock of fruit
flies is blind after elimination of all known photoreceptors. Neuron 30 (1):249-261
243. Helfrich-Förster C, Tauber M, Park JH, Muhlig-Versen M, Schneuwly S, Hofbauer A (2000) Ectopic
expression of the neuropeptide pigment-dispersing factor alters behavioral rhythms in
Drosophila melanogaster. J Neurosci 20 (9):3339-3353
244. Helfrich-Förster C, Shafer OT, Wulbeck C, Grieshaber E, Rieger D, Taghert P (2007a)
Development and morphology of the clock-gene-expressing lateral neurons of Drosophila
melanogaster. J Comp Neurol 500 (1):47-70
245. Helfrich-Förster C, Yoshii T, Wülbeck C, Grieshaber E, Rieger D, Bachleitner W, Cusamano P,
Rouyer F (2007b) The lateral and dorsal neurons of Drosophila melanogaster: new insights
about their morphology and function. Cold Spring Harb Symp Quant Biol 72:517-525
246. Hermes ML, Kolaj M, Doroshenko P, Coderre E, Renaud LP (2009) Effects of VPAC 2 receptor
activation on membrane excitability and GABAergic transmission in subparaventricular zone
neurons targeted by suprachiasmatic nucleus. J Neurophysiol 102 (3):1834-1842
247. Hewes RS (1999) Voltage-dependent ionic currents in the ventromedial eclosion hormone
neurons of Manduca sexta. J Exp Biol 202 (Pt 17):2371-2383
248. Heyer CB, Lux HD (1976) Control of the delayed outward potassium currents in bursting
pacemaker neurons of the snail, Helix pomatia. J Physiol 262 (2):349-382
249. Hida A, Koike N, Hirose M, Hattori M, Sakaki Y, Tei H (2000) The human and mouse period1
genes: five well-conserved E-boxes additively contribute to the enhancement of mPer1
transcription. Genomics 65 (3):224-233
250. Hilgemann DW, Ball R (1996) Regulation of cardiac Na+,Ca2+ exchange and KATP potassium
channels by PIP2. Science 273 (5277):956-959
251. Hill CA, Fox AN, Pitts RJ, Kent LB, Tan PL, Chrystal MA, Cravchik A, Collins FH, Robertson HM,
Zwiebel LJ (2002) G protein-coupled receptors in Anopheles gambiae. Science 298
(5591):176-178
252. Hille B (2001) Ion channels of excitable membranes. third edn. Sinauer Associates, Inc.,
Sunderland, Massachusetts U.S.A.
253. Hodge JJ, Stanewsky R (2008) Function of the Shaw potassium channel within the Drosophila
circadian clock. PLoS One 3 (5):e2274
254. Hofbauer A, Buchner E (1989) Does Drosophila have seven eyes? Naturwissenschaften 76
(7):335-336
255. Hofer S, Homberg U (2006a) Evidence for a role of orcokinin-related peptides in the circadian
clock controlling locomotor activity of the cockroach Leucophaea maderae. Journal of
Experimental Biology 209 (14):2794-2803
252
Bibliography
256. Hofer S, Homberg U (2006b) Orcokinin immunoreactivity in the accessory medulla of the
cockroach Leucophaea maderae. Cell and Tissue Research 325 (3):589-600
257. Holmes AM, Roderick HL, McDonald F, Bootman MD (2007) Interaction between store-operated
and arachidonate-activated calcium entry. Cell Calcium 41 (1):1-12
258. Homberg U, Christensen TA, Hildebrand JG (1989) Structure and function of the deutocerebrum
in insects. Annu Rev Entomol 34:477-501
259. Homberg U, Reischig T, Stengl M (2003) Neural organization of the cireadian system of the
cockroach Leueophaea maderae. Chronobiology International 20 (4):577-591
260. Homberg U, Wurden S, Dircksen H, Rao KR (1991) Comparative anatomy of pigment-dispersing
hormone-immunoreactive neurons in the brain of orthopteroid insects. Cell and Tissue
Research 266 (2):343-357
261. Hong JH, Min CH, Jeong B, Kojiya T, Morioka E, Nagai T, Ikeda M, Lee KJ (2010) Intracellular
calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.
PLoS One 5 (3):e9634
262. Hooper R, Rothberg BS, Soboloff J (2014) Neuronal STIMulation at Rest. Sci Signal 7 (335):pe18
263. Hopf TA, Morinaga S, Ihara S, Touhara K, Marks DS, Benton R (2015) Amino acid coevolution
reveals three-dimensional structure and functional domains of insect odorant receptors. Nat
Commun 6:6077
264. Horn R, Marty A (1988) Muscarinic activation of ionic currents measured by a new whole-cell
recording method. The Journal of General Physiology 92 (2):145-159
265. Houl JH, Yu W, Dudek SM, Hardin PE (2006) Drosophila CLOCK is constitutively expressed in
circadian oscillator and non-oscillator cells. J Biol Rhythms 21 (2):93-103
266. Huang CL, Feng S, Hilgemann DW (1998) Direct activation of inward rectifier potassium channels
by PIP2 and its stabilization by G. Nature 391 (6669):803-806
267. Huang ZJ, Edery I, Rosbash M (1993) PAS is a dimerization domain common to Drosophila period
and several transcription factors. Nature 364 (6434):259-262
268. Hull JJ, Hoffmann EJ, Perera OP, Snodgrass GL (2012) Identification of the western tarnished
plant bug (Lygus hesperus) olfactory co-receptor Orco: Expression profile and confirmation of
atypical membrane topology. Arch Insect Biochem Physiol
269. Hunter-Ensor M, Ousley A, Sehgal A (1996) Regulation of the Drosophila protein timeless
suggests a mechanism for resetting the circadian clock by light. Cell 84 (5):677-685
270. Hyun S, Lee Y, Hong ST, Bang S, Paik D, Kang J, Shin J, Lee J, Jeon K, Hwang S, Bae E, Kim J (2005)
Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF. Neuron 48
(2):267-278
271. Ibata Y, Takahashi Y, Okamura H, Kawakami F, Terubayashi H, Kubo T, Yanaihara N (1989)
Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat
suprachiasmatic nucleus receive a direct retinal projection. Neurosci Lett 97 (1-2):1-5
272. Ignatious Raja JS, Katanayeva N, Katanaev VL, Galizia CG (2014) Role of G o/i subgroup of G
proteins in olfactory signaling of Drosophila melanogaster. Eur J Neurosci
273. Ihara M, Ishida C, Okuda H, Ozoe Y, Matsuda K (2005) Differential blocking actions of 4'-ethynyl4-n-propylbicycloorthobenzoate (EBOB) and -hexachlorocyclohexane (-HCH) on aminobutyric acid- and glutamate-induced responses of American cockroach neurons. Invert
Neurosci 5 (3-4):157-164
274. Ikeda T, Zhao X, Kono Y, Yeh JZ, Narahashi T (2003) Fipronil modulation of glutamate-induced
chloride currents in cockroach thoracic ganglion neurons. Neurotoxicology 24 (6):807-815
275. Im SH, Taghert PH (2010) PDF receptor expression reveals direct interactions between circadian
oscillators in Drosophila. J Comp Neurol 518 (11):1925-1945
276. Im SH, Li W, Taghert PH (2011) PDFR and CRY signaling converge in a subset of clock neurons to
modulate the amplitude and phase of circadian behavior in Drosophila. PLoS One 6
(4):e18974
277. Immonen E, Ritchie MG (2012) The genomic response to courtship song stimulation in female
Drosophila melanogaster. Proc Biol Sci 279 (1732):1359-1365
253
Bibliography
278. Isaac RE, Johnson EC, Audsley N, Shirras AD (2007) Metabolic inactivation of the circadian
transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila. J Exp
Biol 210 (Pt 24):4465-4470
279. Ishihara T, Shigemoto R, Mori K, Takahashi K, Nagata S (1992) Functional expression and tissue
distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron 8 (4):811-819
280. Itagaki H, Conner WE (1988) Calling behavior of Manduca sexta (L.) (Lepidoptera: Sphingidae)
with notes on the morphology of the female sex pheromone gland. Annals of the
Entomological Society of America 81 (5):798-807
281. Ito C, Goto SG, Shiga S, Tomioka K, Numata H (2008) Peripheral circadian clock for the cuticle
deposition rhythm in Drosophila melanogaster. Proc Natl Acad Sci U S A 105 (24):8446-8451
282. Itri J, Colwell CS (2003) Regulation of inhibitory synaptic transmission by vasoactive intestinal
peptide (VIP) in the mouse suprachiasmatic nucleus. J Neurophysiol 90 (3):1589-1597
283. Ivanchenko M, Stanewsky R, Giebultowicz JM (2001) Circadian photoreception in Drosophila:
functions of cryptochrome in peripheral and central clocks. J Biol Rhythms 16 (3):205-215
284. Ivens I, Deitmer JW (1986) Inhibition of a voltage-dependent Ca2+ current by concanavalin A.
Pflugers Arch 406 (2):212-217
285. Iwai S, Fukui Y, Fujiwara Y, Takeda M (2006) Structure and expressions of two circadian clock
genes, period and timeless in the commercial silkmoth, Bombyx mori. J Insect Physiol 52
(6):625-637
286. Izhikevich EM, Desai NS, Walcott EC, Hoppensteadt FC (2003) Bursts as a unit of neural
information: selective communication via resonance. Trends Neurosci 26 (3):161-167
287. Jackson FR (2011) Glial cell modulation of circadian rhythms. Glia 59 (9):1341-1350
288. Jacquin-Joly E, Francois MC, Burnet M, Lucas P, Bourrat F, Maida R (2002) Expression pattern in
the antennae of a newly isolated lepidopteran Gq protein α subunit cDNA. Eur J Biochem 269
(8):2133-2142
289. Janssen D, Derst C, Buckinx R, Van den Eynden J, Rigo JM, Van Kerkhove E (2007) Dorsal
unpaired median neurons of Locusta migratoria express ivermectin- and fipronil-sensitive
glutamate-gated chloride channels. J Neurophysiol 97 (4):2642-2650
290. Jaramillo AM, Zheng X, Zhou Y, Amado DA, Sheldon A, Sehgal A, Levitan IB (2004) Pattern of
distribution and cycling of SLOB, Slowpoke channel binding protein, in Drosophila. BMC
Neurosci 5:3
291. Jin X, Ha TS, Smith DP (2008) SNMP is a signaling component required for pheromone sensitivity
in Drosophila. Proc Natl Acad Sci U S A 105 (31):10996-11001
292. Jindra M, Huang JY, Malone F, Asahina M, Riddiford LM (1997) Identification and mRNA
developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca
sexta. Insect Mol Biol 6 (1):41-53
293. Johard HA, Yoishii T, Dircksen H, Cusumano P, Rouyer F, Helfrich-Förster C, Nässel DR (2009)
Peptidergic clock neurons in Drosophila: ion transport peptide and short neuropeptide F in
subsets of dorsal and ventral lateral neurons. J Comp Neurol 516 (1):59-73
294. Johnson CH, Golden SS, Ishiura M, Kondo T (1996) Circadian clocks in prokaryotes. Mol
Microbiol 21 (1):5-11
295. Jones PL, Pask GM, Rinker DC, Zwiebel LJ (2011) Functional agonism of insect odorant receptor
ion channels. Proc Natl Acad Sci U S A 108 (21):8821-8825
296. Jones PL, Pask GM, Romaine IM, Taylor RW, Reid PR, Waterson AG, Sulikowski GA, Zwiebel LJ
(2012) Allosteric antagonism of insect odorant receptor ion channels. PLoS One 7 (1):e30304
297. Jones WD, Nguyen TA, Kloss B, Lee KJ, Vosshall LB (2005) Functional conservation of an insect
odorant receptor gene across 250 million years of evolution. Curr Biol 15 (4):R119-R121
298. Jordan MD, Anderson A, Begum D, Carraher C, Authier A, Marshall SD, Kiely A, Gatehouse LN,
Greenwood DR, Christie DL, Kralicek AV, Trowell SC, Newcomb RD (2009) Odorant receptors
from the light brown apple moth (Epiphyas postvittana) recognize important volatile
compounds produced by plants. Chem Senses 34 (5):383-394
254
Bibliography
299. Kadener S, Stoleru D, McDonald M, Nawathean P, Rosbash M (2007) Clockwork Orange is a
transcriptional repressor and a new Drosophila circadian pacemaker component. Genes Dev
21 (13):1675-1686
300. Kain P, Chakraborty TS, Sundaram S, Siddiqi O, Rodrigues V, Hasan G (2008) Reduced odor
responses from antennal neurons of Gqα, phospholipase Cβ, and rdgA mutants in Drosophila
support a role for a phospholipid intermediate in insect olfactory transduction. J Neurosci 28
(18):4745-4755
301. Kaissling KE (1986) Chemo-electrical transduction in insect olfactory receptors. Annu Rev
Neurosci 9:121-145
302. Kaissling KE (2009) Olfactory perireceptor and receptor events in moths: a kinetic model revised.
J Comp Physiol A Neuroethol Sens Neural Behav Physiol 195 (10):895-922
303. Kaissling KE (2013) Kinetics of olfactory responses might largely depend on the odorant-receptor
interaction and the odorant deactivation postulated for flux detectors. J Comp Physiol A
Neuroethol Sens Neural Behav Physiol
304. Kaissling KE, Priesner E (1970) Die Riechschwelle des Seidenspinners. Naturwissenschaften 57
(1):23-28
305. Kaissling KE, Hildebrand JG, Tumlinson JH (1989) Pheromone receptor-cells in the male moth
Manduca sexta. Archives of Insect Biochemistry and Physiology 10 (4):273-279
306. Kalidas S, Smith DP (2002) Novel genomic cDNA hybrids produce effective RNA interference in
adult Drosophila. Neuron 33 (2):177-184
307. Kalinova B, Hoskovec M, Liblikas I, Unelius CR, Hansson BS (2001) Detection of sex pheromone
components in Manduca sexta (L.). Chem Senses 26 (9):1175-1186
308. Kallo I, Kalamatianos T, Piggins HD, Coen CW (2004a) Ageing and the diurnal expression of
mRNAs for vasoactive intestinal peptide and for the VPAC2 and PAC1 receptors in the
suprachiasmatic nucleus of male rats. J Neuroendocrinol 16 (9):758-766
309. Kallo I, Kalamatianos T, Wiltshire N, Shen S, Sheward WJ, Harmar AJ, Coen CW (2004b)
Transgenic approach reveals expression of the VPAC2 receptor in phenotypically defined
neurons in the mouse suprachiasmatic nucleus and in its efferent target sites. Eur J Neurosci
19 (8):2201-2211
310. Kameyama A, Yazawa K, Kaibara M, Ozono K, Kameyama M (1997) Run-down of the cardiac Ca2+
channel: characterization and restoration of channel activity by cytoplasmic factors. Pflugers
Arch 433 (5):547-556
311. Kameyama M, Kameyama A, Takano E, Maki M (1998) Run-down of the cardiac L-type Ca2+
channel: partial restoration of channel activity in cell-free patches by calpastatin. Pflugers
Arch 435 (3):344-349
312. Kaneko M, Hall JC (2000) Neuroanatomy of cells expressing clock genes in Drosophila: transgenic
manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker
neurons and their projections. J Comp Neurol 422 (1):66-94
313. Kaneko M, Helfrich-Förster C, Hall JC (1997) Spatial and temporal expression of the period and
timeless genes in the developing nervous system of Drosophila: newly identified pacemaker
candidates and novel features of clock gene product cycling. J Neurosci 17 (17):6745-6760
314. Kang GJ, Gong ZJ, Cheng JA, Zhu ZR, Mao CG (2011) Cloning and expression analysis of a Gprotein  subunit - Go in the rice water weevil Lissorhoptrus oryzophilus Kuschel. Arch Insect
Biochem Physiol 76 (1):43-54
315. Keil TA (1989) Fine structure of the pheromone-sensitive sensilla on the antenna of the
hawkmoth, Manduca sexta. Tissue Cell 21 (1):139-151
316. Kepplinger KJF, Romanin C (2005) The run-down phenomenon of Ca2+ channels. In: Zamponi G
(ed) Voltage-gated calcium channels. Eurekah.com and Kluwer Academic / Plenum
Publishers, pp 219-230
317. Khazipov R, Minlebaev M, Valeeva G (2013) Early gamma oscillations. Neuroscience 250:240252
255
Bibliography
318. Kiely A (2008) Functional and structural analyses of an olfactory receptor from Drosophila
melanogaster. Dissertation, University of Auckland,
319. Kiely A, Authier A, Kralicek AV, Warr CG, Newcomb RD (2007) Functional analysis of a Drosophila
melanogaster olfactory receptor expressed in Sf9 cells. J Neurosci Methods 159 (2):189-194
320. Kim EY, Edery I (2006) Balance between DBT/CKIε kinase and protein phosphatase activities
regulate phosphorylation and stability of Drosophila CLOCK protein. Proc Natl Acad Sci U S A
103 (16):6178-6183
321. Kim WJ, Jan LY, Jan YN (2013) A PDF/NPF neuropeptide signaling circuitry of male Drosophila
melanogaster controls rival-induced prolonged mating. Neuron 80 (5):1190-1205
322. Klarsfeld A, Malpel S, Michard-Vanhee C, Picot M, Chelot E, Rouyer F (2004) Novel features of
Cryptochrome-mediated photoreception in the brain circadian clock of Drosophila. J Neurosci
24 (6):1468-1477
323. Klein IB, Kirsch JF (1969) The activation of papain and the inhibition of the active enzyme by
carbonyl reagents. J Biol Chem 244 (21):5928-5935
324. Klein U (1987) Sensillum-lymph proteins from antennal olfactory hairs of the moth Antheraea
polyphemus (Saturniidae). Insect Biochemistry 17 (8):1193-1204
325. Klevecz RR, Pilliod J, Bolen J (1991) Autogenous formation of spiral waves by coupled chaotic
attractors. Chronobiol Int 8 (1):6-13
326. Kleyman TR, Cragoe EJ, Jr. (1988) Amiloride and its analogs as tools in the study of ion transport.
J Membr Biol 105 (1):1-21
327. Kloss B, Rothenfluh A, Young MW, Saez L (2001) Phosphorylation of PERIOD is influenced by
cycling physical associations of DOUBLE-TIME, PERIOD, and TIMELESS in the Drosophila clock.
Neuron 30 (3):699-706
328. Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Young MW (1998) The Drosophila clock
gene double-time encodes a protein closely related to human casein kinase Iε. Cell 94 (1):97107
329. Knowles A, Koh K, Wu JT, Chien CT, Chamovitz DA, Blau J (2009) The COP9 signalosome is
required for light-dependent Timeless degradation and Drosophila clock resetting. J Neurosci
29 (4):1152-1162
330. Ko HW, Jiang J, Edery I (2002) Role for Slimb in the degradation of Drosophila Period protein
phosphorylated by Doubletime. Nature 420 (6916):673-678
331. Koh K, Zheng X, Sehgal A (2006) JETLAG resets the Drosophila circadian clock by promoting lightinduced degradation of TIMELESS. Science 312 (5781):1809-1812
332. Kononenko NI, Shao LR, Dudek FE (2004) Riluzole-sensitive slowly inactivating sodium current in
rat suprachiasmatic nucleus neurons. J Neurophysiol 91 (2):710-718
333. Konopka RJ, Benzer S (1971) Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A
68 (9):2112-2116
334. Konopka RJ, Pittendrigh C, Orr D (1989) Reciprocal behaviour associated with altered
homeostasis and photosensitivity of Drosophila clock mutants. J Neurogenet 6 (1):1-10
335. Körte S (2013) Pharmacological characterization of the odorant receptor MsextaOR-2. Diploma
thesis, University of Kassel, Kassel
336. Kostyuk PG, Veselovsky NS, Fedulova SA (1981) Ionic currents in the somatic membrane of rat
dorsal root ganglion neurons - II. Calcium currents. Neuroscience 6 (12):2431-2437
337. Kragh-Hansen U (1981) Molecular aspects of ligand binding to serum albumin. Pharmacol Rev 33
(1):17-53
338. Krannich S (2008) Electrophysiological and pharmacological characterization of ion channels
involved in moth olfactory transduction cascades. PhD-thesis, Philipps University, Marburg
339. Krannich S, Stengl M (2008) Cyclic nucleotide-activated currents in cultured olfactory receptor
neurons of the hawkmoth Manduca sexta. J Neurophysiol 100 (5):2866-2877
340. Kreissl S, Bicker G (1992) Dissociated neurons of the pupal honeybee brain in cell culture. J
Neurocytol 21 (8):545-556
256
Bibliography
341. Krieger J, Klink O, Mohl C, Raming K, Breer H (2003) A candidate olfactory receptor subtype
highly conserved across different insect orders. J Comp Physiol A Neuroethol Sens Neural
Behav Physiol 189 (7):519-526
342. Krieger J, Raming K, Dewer YM, Bette S, Conzelmann S, Breer H (2002) A divergent gene family
encoding candidate olfactory receptors of the moth Heliothis virescens. Eur J Neurosci 16
(4):619-628
343. Krishnan B, Dryer SE, Hardin PE (1999) Circadian rhythms in olfactory responses of Drosophila
melanogaster. Nature 400 (6742):375-378
344. Krishnan B, Levine JD, Lynch MK, Dowse HB, Funes P, Hall JC, Hardin PE, Dryer SE (2001) A new
role for cryptochrome in a Drosophila circadian oscillator. Nature 411 (6835):313-317
345. Krishnan P, Dryer SE, Hardin PE (2005) Measuring circadian rhythms in olfaction using
electroantennograms. Methods Enzymol 393:495-508
346. Krishnan P, Chatterjee A, Tanoue S, Hardin PE (2008) Spike amplitude of single-unit responses in
antennal sensillae is controlled by the Drosophila circadian clock. Curr Biol 18 (11):803-807
347. Krsmanovic LZ, Mores N, Navarro CE, Arora KK, Catt KJ (2003) An agonist-induced switch in G
protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile
neuropeptide secretion. Proc Natl Acad Sci U S A 100 (5):2969-2974
348. Krupp JJ, Billeter JC, Wong A, Choi C, Nitabach MN, Levine JD (2013) Pigment-dispersing factor
modulates pheromone production in clock cells that influence mating in Drosophila. Neuron
79 (1):54-68
349. Krupp JJ, Kent C, Billeter JC, Azanchi R, So AK, Schonfeld JA, Smith BP, Lucas C, Levine JD (2008)
Social experience modifies pheromone expression and mating behavior in male Drosophila
melanogaster. Curr Biol 18 (18):1373-1383
350. Kudo T, Tahara Y, Gamble KL, McMahon DG, Block GD, Colwell CS (2013) Vasoactive intestinal
peptide produces long-lasting changes in neural activity in the suprachiasmatic nucleus. J
351. Kula-Eversole E, Nagoshi E, Shang Y, Rodriguez J, Allada R, Rosbash M (2010) Surprising gene
expression patterns within and between PDF-containing circadian neurons in Drosophila.
352. Kula E, Levitan ES, Pyza E, Rosbash M (2006) PDF cycling in the dorsal protocerebrum of the
Drosophila brain is not necessary for circadian clock function. J Biol Rhythms 21 (2):104-117
353. Kumar BN, Taylor RW, Pask GM, Zwiebel LJ, Newcomb RD, Christie DL (2013) A conserved
aspartic acid is important for agonist (VUAA1) and odorant/tuning receptor-dependent
activation of the insect odorant co-receptor (Orco). PLoS One 8 (7):e70218
354. Kurtovic A, Widmer A, Dickson BJ (2007) A single class of olfactory neurons mediates
behavioural responses to a Drosophila sex pheromone. Nature 446 (7135):542-546
355. Kwon HW, Lu T, Rutzler M, Zwiebel LJ (2006) Olfactory responses in a gustatory organ of the
malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci U S A 103 (36):13526-13531
356. Kwon JY, Dahanukar A, Weiss LA, Carlson JR (2007) The molecular basis of CO2 reception in
Drosophila. Proc Natl Acad Sci U S A 104 (9):3574-3578
357. Kyrozis A, Reichling DB (1995) Perforated-patch recording with gramicidin avoids artifactual
changes in intracellular chloride concentration. J Neurosci Methods 57 (1):27-35
358. Lacaille F, Hiroi M, Twele R, Inoshita T, Umemoto D, Maniere G, Marion-Poll F, Ozaki M, Francke
W, Cobb M, Everaerts C, Tanimura T, Ferveur JF (2007) An inhibitory sex pheromone tastes
bitter for Drosophila males. PLoS One 2 (7):e661
359. Lapied B, Macélot CO, Pelhate M (1990) Patch-clamp study of the properties of the sodium
current in cockroach single isolated adult aminergic neurones. Journal of Experimental
Biology 151 (1):387-403
360. Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, Vosshall LB (2004) Or83b encodes
a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43 (5):703714
257
Bibliography
361. Laue M, Steinbrecht RA (1997) Topochemistry of moth olfactory sensilla. International Journal of
Insect Morphology and Embryology 26 (3–4):217-228
362. Laue M, Maida R, Redkozubov A (1997) G-protein activation, identification and
immunolocalization in pheromone-sensitive sensilla trichodea of moths. Cell Tissue Res 288
(1):149-158
363. Lear BC, Zhang L, Allada R (2009) The neuropeptide PDF acts directly on evening pacemaker
neurons to regulate multiple features of circadian behavior. PLoS Biol 7 (7):e1000154
364. Lear BC, Lin JM, Keath JR, McGill JJ, Raman IM, Allada R (2005a) The ion channel Narrow
Abdomen is critical for neural output of the Drosophila circadian pacemaker. Neuron 48
(6):965-976
365. Lear BC, Merrill CE, Lin JM, Schroeder A, Zhang L, Allada R (2005b) A G protein-coupled receptor,
groom-of-PDF, is required for PDF neuron action in circadian behavior. Neuron 48 (2):221227
366. Lee C, Bae K, Edery I (1998) The Drosophila CLOCK protein undergoes daily rhythms in
abundance, phosphorylation, and interactions with the PER-TIM complex. Neuron 21 (4):857867
367. Lee C, Bae K, Edery I (1999) PER and TIM inhibit the DNA binding activity of a Drosophila CLOCKCYC/dBMAL1 heterodimer without disrupting formation of the heterodimer: a basis for
circadian transcription. Mol Cell Biol 19 (8):5316-5325
368. Lee CM, Su MT, Lee HJ (2009) Pigment dispersing factor: an output regulator of the circadian
clock in the German cockroach. J Biol Rhythms 24 (1):35-43
369. Lee JK, Strausfeld NJ (1990) Structure, distribution and number of surface sensilla and their
receptor cells on the olfactory appendage of the male moth Manduca sexta. J Neurocytol 19
(4):519-538
370. Lee Y, Kim SH, Montell C (2010) Avoiding DEET through insect gustatory receptors. Neuron 67
(4):555-561
371. Lehman HK Circadian control of Manduca sexta flight. In: Abstr Soc Neurosci, 1990. p 1334
372. Lelito KR, Shafer OT (2012) Reciprocal cholinergic and GABAergic modulation of the small
ventrolateral pacemaker neurons of Drosophila's circadian clock neuron network. J
373. Lemon W, Getz W (1997) Temporal resolution of general odor pulses by olfactory sensory
neurons in American cockroaches. J Exp Biol 200 (Pt 12):1809-1819
374. Leung HT, Tseng-Crank J, Kim E, Mahapatra C, Shino S, Zhou Y, An L, Doerge RW, Pak WL (2008)
DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors.
Neuron 58 (6):884-896
375. Levine JD, Funes P, Dowse HB, Hall JC (2002a) Resetting the circadian clock by social experience
in Drosophila melanogaster. Science 298 (5600):2010-2012
376. Levine JD, Funes P, Dowse HB, Hall JC (2002b) Advanced analysis of a cryptochrome mutation's
effects on the robustness and phase of molecular cycles in isolated peripheral tissues of
Drosophila. BMC Neurosci 3:5
377. Levitan IB (1994) Modulation of ion channels by protein phosphorylation and
dephosphorylation. Annu Rev Physiol 56:193-212
378. Lewcock JW, Reed RR (2004) A feedback mechanism regulates monoallelic odorant receptor
expression. Proc Natl Acad Sci U S A 101 (4):1069-1074
379. Li KM, Ren LY, Zhang YJ, Wu KM, Guo YY (2012) Knockdown of Microplitis mediator odorant
receptor involved in the sensitive detection of two chemicals. J Chem Ecol 38 (3):287-294
380. Li M, West JW, Lai Y, Scheuer T, Catterall WA (1992) Functional modulation of brain sodium
channels by cAMP-dependent phosphorylation. Neuron 8 (6):1151-1159
381. Li Y, Guo F, Shen J, Rosbash M (2014a) PDF and cAMP enhance PER stability in Drosophila clock
neurons. Proc Natl Acad Sci U S A 111 (13):E1284-1290
382. Li Z, Ni JD, Huang J, Montell C (2014b) Requirement for Drosophila SNMP1 for rapid activation
and termination of pheromone-induced activity. PLoS Genet 10 (9):e1004600
258
Bibliography
383. Lim C, Chung BY, Pitman JL, McGill JJ, Pradhan S, Lee J, Keegan KP, Choe J, Allada R (2007)
Clockwork orange encodes a transcriptional repressor important for circadian-clock
amplitude in Drosophila. Curr Biol 17 (12):1082-1089
384. Lim C, Lee J, Choi C, Kilman VL, Kim J, Park SM, Jang SK, Allada R, Choe J (2011) The novel gene
twenty-four defines a critical translational step in the Drosophila clock. Nature 470
(7334):399-403
385. Lin FJ, Song W, Meyer-Bernstein E, Naidoo N, Sehgal A (2001) Photic signaling by Cryptochrome
in the Drosophila circadian system. Mol Cell Biol 21 (21):7287-7294
386. Lin GG, Liou RF, Lee HJ (2002a) The period gene of the German cockroach and its novel linking
power between vertebrate and invertebrate. Chronobiol Int 19 (6):1023-1040
387. Lin JM, Schroeder A, Allada R (2005) In vivo circadian function of casein kinase 2
phosphorylation sites in Drosophila PERIOD. J Neurosci 25 (48):11175-11183
388. Lin JM, Kilman VL, Keegan K, Paddock B, Emery-Le M, Rosbash M, Allada R (2002b) A role for
casein kinase 2α in the Drosophila circadian clock. Nature 420 (6917):816-820
389. Lin Y, Stormo GD, Taghert PH (2004) The neuropeptide pigment-dispersing factor coordinates
pacemaker interactions in the Drosophila circadian system. J Neurosci 24 (36):7951-7957
390. Lindau M, Fernandez JM (1986) IgE-mediated degranulation of mast cells does not require
opening of ion channels. Nature 319 (6049):150-153
391. Lingren PD, Greene GL, Davis DR, Baumhover AH, Henneberry TJ (1977) Nocturnal behavior of
four lepidopteran pests that attack tobacco and other crops. Annals of the Entomological
Society of America 70 (2):161-167
392. Linn CE, Campbell MG, Poole KR, Wen-Q W, Roelofs WL (1996) Effects of photoperiod on the
circadian timing of pheromone response in male Trichoplusia ni: relationship to the
modulatory action of octopamine. Journal of Insect Physiology 42 (9):881-891
393. Linn Jr CE, Poole KR, Roelofs WL (1994a) Studies on biogenic amines and their metabolites in
nervous tissue and hemolymph of adult male cabbage looper moths - I. Quantitation of
photoperiod changes. Comparative Biochemistry and Physiology Part C: Pharmacology,
Toxicology and Endocrinology 108 (1):73-85
394. Linn Jr CE, Campbell MG, Poole KR, Roelofs WL (1994b) Studies on biogenic amines and their
metabolites in nervous tissue and hemolymph of male cabbage looper moths - II.
Photoperiod changes relative to random locomotor activity and pheromone-response
thresholds. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and
Endocrinology 108 (1):87-98
395. Linnaeus C (1763) Centuria insectorum (proposuit Boas Johansson). In: Amoenitates
academicae.
396. Lischka FW, Schild D (1993) Standing calcium gradients in olfactory receptor neurons can be
abolished by amiloride or ruthenium red. J Gen Physiol 102 (5):817-831
397. Lisman JE (1997) Bursts as a unit of neural information: making unreliable synapses reliable.
Trends Neurosci 20 (1):38-43
398. Littleton JT, Ganetzky B (2000) Ion channels and synaptic organization: analysis of the
Drosophila genome. Neuron 26 (1):35-43
399. Liu C, Reppert SM (2000) GABA synchronizes clock cells within the suprachiasmatic circadian
clock. Neuron 25 (1):123-128
400. Liu C, Zhang J, Liu Y, Wang G, Dong S (2014a) Expression of snmp1 and snmp2 genes in antennal
sensilla of Spodoptera exigua (Hübner). Arch Insect Biochem Physiol
401. Liu C, Pitts RJ, Bohbot JD, Jones PL, Wang G, Zwiebel LJ (2010) Distinct olfactory signaling
mechanisms in the malaria vector mosquito Anopheles gambiae. PLoS Biol 8 (8)
402. Liu WW, Wilson RI (2013) Glutamate is an inhibitory neurotransmitter in the Drosophila
olfactory system. Proc Natl Acad Sci U S A 110 (25):10294-10299
403. Liu X, Lorenz L, Yu QN, Hall JC, Rosbash M (1988) Spatial and temporal expression of the period
gene in Drosophila melanogaster. Genes Dev 2 (2):228-238
259
Bibliography
404. Liu YC, Pearce MW, Honda T, Johnson TK, Charlu S, Sharma KR, Imad M, Burke RE, Zinsmaier KE,
Ray A, Dahanukar A, de Bruyne M, Warr CG (2014b) The Drosophila melanogaster
phospholipid flippase dATP8B is required for odorant receptor function. PLoS Genet 10
(3):e1004209
405. Loesel R, Homberg U (1998) Sustained oscillations in an insect visual system.
Naturwissenschaften 85 (5):238-240
406. Loesel R, Homberg U (1999) Histamine-immunoreactive neurons in the brain of the cockroach
Leucophaea maderae. Brain Res 842 (2):408-418
407. Loesel R, Homberg U (2001) Anatomy and physiology of neurons with processes in the accessory
medulla of the cockroach Leucophaea maderae. J Comp Neurol 439 (2):193-207
408. Loher W (1972) Circadian control of stridulation in the cricket Teleogryllus commodus Walker. J
Comp Physiol 79 (2):173-190
409. Lone SR, Sharma VK (2011) Social synchronization of circadian locomotor activity rhythm in the
fruit fly Drosophila melanogaster. J Exp Biol 214 (Pt 22):3742-3750
410. Lone SR, Sharma VK (2012) Or47b receptor neurons mediate sociosexual interactions in the fruit
fly Drosophila melanogaster. J Biol Rhythms 27 (2):107-116
411. Long MA, Jutras MJ, Connors BW, Burwell RD (2005) Electrical synapses coordinate activity in
the suprachiasmatic nucleus. Nat Neurosci 8 (1):61-66
412. Loudon C, Koehl MA (2000) Sniffing by a silkworm moth: wing fanning enhances air penetration
through and pheromone interception by antennae. J Exp Biol 203 (19):2977-2990
413. Lu B, Wang N, Xiao J, Xu Y, Murphy RW, Huang D (2009) Expression and evolutionary divergence
of the non-conventional olfactory receptor in four species of fig wasp associated with one
species of fig. BMC Evol Biol 9:43
414. Lucas P, Shimahara T (2002) Voltage- and calcium-activated currents in cultured olfactory
receptor neurons of male Mamestra brassicae (Lepidoptera). Chem Senses 27 (7):599-610
415. Lundin C, Kall L, Kreher SA, Kapp K, Sonnhammer EL, Carlson JR, Heijne G, Nilsson I (2007)
Membrane topology of the Drosophila OR83b odorant receptor. FEBS Lett 581 (29):56015604
416. Lutz EM, Sheward WJ, West KM, Morrow JA, Fink G, Harmar AJ (1993) The VIP 2 receptor:
molecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal
peptide. FEBS Lett 334 (1):3-8
417. Ma L, Gu SH, Liu ZW, Wang SN, Guo YY, Zhou JJ, Zhang YJ (2013) Molecular characterization and
expression profiles of olfactory receptor genes in the parasitic wasp, Microplitis mediator
(Hymenoptera: Braconidae). J Insect Physiol 60C:118-126
418. Maida R, Redkozubov A, Ziegelberger G (2000) Identification of PLC β and PKC in pheromone
receptor neurons of Antheraea polyphemus. Neuroreport 11 (8):1773-1776
419. Manzini I, Schild D (2003) Multidrug resistance transporters in the olfactory receptor neurons of
Xenopus laevis tadpoles. J Physiol 546 (Pt 2):375-385
420. Manzini I, Schweer TS, Schild D (2008) Improved fluorescent (calcium indicator) dye uptake in
brain slices by blocking multidrug resistance transporters. J Neurosci Methods 167 (2):140147
421. Marder E (2012) Neuromodulation of neuronal circuits: back to the future. Neuron 76 (1):1-11
422. Marder E, Richards KS (1999) Development of the peptidergic modulation of a rhythmic pattern
generating network. Brain Res 848 (1-2):35-44
423. Marion-Poll F, Tobin TR (1992) Temporal coding of pheromone pulses and trains in Manduca
sexta. J Comp Physiol A 171 (4):505-512
424. Martin F, Charro MJ, Alcorta E (2001) Mutations affecting the cAMP transduction pathway
modify olfaction in Drosophila. J Comp Physiol A 187 (5):359-370
425. Martinek S, Inonog S, Manoukian AS, Young MW (2001) A role for the segment polarity gene
shaggy/GSK-3 in the Drosophila circadian clock. Cell 105 (6):769-779
426. Marty A, Finkelstein A (1975) Pores formed in lipid bilayer membranes by nystatin - Differences
in its one-sided and two-sided action. J Gen Physiol 65 (4):515-526
260
Bibliography
427. Matsumoto A, Ukai-Tadenuma M, Yamada RG, Houl J, Uno KD, Kasukawa T, Dauwalder B, Itoh
TQ, Takahashi K, Ueda R, Hardin PE, Tanimura T, Ueda HR (2007) A functional genomics
strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian
clock. Genes Dev 21 (13):1687-1700
428. Matsushima A, Sato S, Chuman Y, Takeda Y, Yokotani S, Nose T, Tominaga Y, Shimohigashi M,
Shimohigashi Y (2004) cDNA cloning of the housefly pigment-dispersing factor (PDF)
precursor protein and its peptide comparison among the insect circadian neuropeptides. J
Pept Sci 10 (2):82-91
429. Matsushima A, Yokotani S, Lui X, Sumida K, Honda T, Sato S, Kaneki A, Takeda Y, Chuman Y,
Ozaki M, Asai D, Nose T, Onoue H, Ito Y, Tominaga Y, Shimohigashi Y, Shimohigashi M (2003)
Molecular cloning and circadian expression profile of insect neuropeptide PDF in black
blowfly, Phormia regina. Int J Pept Res Ther 10 (5-6):419-430
430. Maywood ES, Reddy AB, Wong GK, O'Neill JS, O'Brien JA, McMahon DG, Harmar AJ, Okamura H,
Hastings MH (2006) Synchronization and maintenance of timekeeping in suprachiasmatic
circadian clock cells by neuropeptidergic signaling. Curr Biol 16 (6):599-605
431. Mazzoni EO, Desplan C, Blau J (2005) Circadian pacemaker neurons transmit and modulate
visual information to control a rapid behavioral response. Neuron 45 (2):293-300
432. McCarthy EV, Wu Y, Decarvalho T, Brandt C, Cao G, Nitabach MN (2011) Synchronized bilateral
synaptic inputs to Drosophila melanogaster neuropeptidergic rest/arousal neurons. J
Neurosci 31 (22):8181-8193
433. McDonald MJ, Rosbash M (2001) Microarray analysis and organization of circadian gene
expression in Drosophila. Cell 107 (5):567-578
434. Meelkop E, Temmerman L, Schoofs L, Janssen T (2011) Signalling through pigment dispersing
hormone-like peptides in invertebrates. Prog Neurobiol 93 (1):125-147
435. Meissner RA, Kilman VL, Lin JM, Allada R (2008) TIMELESS is an important mediator of CK2
effects on circadian clock function in vivo. J Neurosci 28 (39):9732-9740
436. Melo AC, Rutzler M, Pitts RJ, Zwiebel LJ (2004) Identification of a chemosensory receptor from
the yellow fever mosquito, Aedes aegypti, that is highly conserved and expressed in olfactory
and gustatory organs. Chem Senses 29 (5):403-410
437. Merker B (2013) Cortical gamma oscillations: the functional key is activation, not cognition.
Neurosci Biobehav Rev 37 (3):401-417
438. Merlin C, Gegear RJ, Reppert SM (2009) Antennal circadian clocks coordinate sun compass
orientation in migratory monarch butterflies. Science 325 (5948):1700-1704
439. Merlin C, Francois MC, Queguiner I, Maibeche-Coisne M, Jacquin-Joly E (2006) Evidence for a
putative antennal clock in Mamestra brassicae: molecular cloning and characterization of
two clock genes - period and cryptochrome - in antennae. Insect Mol Biol 15 (2):137-145
440. Merlin C, Lucas P, Rochat D, Francois MC, Maibeche-Coisne M, Jacquin-Joly E (2007) An antennal
circadian clock and circadian rhythms in peripheral pheromone reception in the moth
Spodoptera littoralis. J Biol Rhythms 22 (6):502-514
441. Mertens I, Vandingenen A, Johnson EC, Shafer OT, Li W, Trigg JS, De Loof A, Schoofs L, Taghert
PH (2005) PDF receptor signaling in Drosophila contributes to both circadian and geotactic
behaviors. Neuron 48 (2):213-219
442. Meves H (2008) Arachidonic acid and ion channels: an update. Br J Pharmacol 155 (1):4-16
443. Meyer-Spasche A, Piggins HD (2004) Vasoactive intestinal polypeptide phase-advances the rat
suprachiasmatic nuclei circadian pacemaker in vitro via protein kinase A and mitogenactivated protein kinase. Neurosci Lett 358 (2):91-94
444. Meyer P, Saez L, Young MW (2006) PER-TIM interactions in living Drosophila cells: an interval
timer for the circadian clock. Science 311 (5758):226-229
445. Miller R, Tu Z (2008) Odorant receptor c-terminal motifs in divergent insect species. J Insect Sci
8:53
261
Bibliography
446. Mills JD, Pitman RM (1999) Contribution of potassium conductances to a time-dependent
transition in electrical properties of a cockroach motoneuron soma. J Neurophysiol 81
(5):2253-2266
447. Miskiewicz K, Pyza E, Schurmann FW (2004) Ultrastructural characteristics of circadian
pacemaker neurones, immunoreactive to an antibody against a pigment-dispersing hormone
in the fly's brain. Neurosci Lett 363 (1):73-77
448. Missbach C, Dweck HK, Vogel H, Vilcinskas A, Stensmyr MC, Hansson BS, Grosse-Wilde E (2014)
Evolution of insect olfactory receptors. Elife 3:e02115
449. Mitsuno H, Sakurai T, Murai M, Yasuda T, Kugimiya S, Ozawa R, Toyohara H, Takabayashi J,
Miyoshi H, Nishioka T (2008) Identification of receptors of main sex-pheromone components
of three Lepidopteran species. Eur J Neurosci 28 (5):893-902
450. Miura N, Atsumi S, Tabunoki H, Sato R (2005) Expression and localization of three G protein
alpha subunits, Go, Gq, and Gs, in adult antennae of the silkmoth (Bombyx mori). J Comp
Neurol 485 (2):143-152
451. Miura N, Nakagawa T, Touhara K, Ishikawa Y (2010) Broadly and narrowly tuned odorant
receptors are involved in female sex pheromone reception in Ostrinia moths. Insect Biochem
Mol Biol 40 (1):64-73
452. Miyasako Y, Umezaki Y, Tomioka K (2007) Separate sets of cerebral clock neurons are
responsible for light and temperature entrainment of Drosophila circadian locomotor
rhythms. J Biol Rhythms 22 (2):115-126
453. Miyazaki S-i (1980) The ionic mechanism of action potentials in neurosecretory cells and nonneurosecretory cells of the silkworm. J Comp Physiol 140 (1):43-52
454. Mizrak D, Ruben M, Myers GN, Rhrissorrakrai K, Gunsalus KC, Blau J (2012) Electrical activity can
impose time of day on the circadian transcriptome of pacemaker neurons. Curr Biol 22
(20):1871-1880
455. Moore RY, Speh JC (1993) GABA is the principal neurotransmitter of the circadian system.
Neurosci Lett 150 (1):112-116
456. Moreaux L, Laurent G (2007) Estimating firing rates from calcium signals in locust projection
neurons in vivo. Front Neural Circuits 1:2
457. Moreaux L, Laurent G (2008) A simple method to reconstruct firing rates from dendritic calcium
signals. Front Neurosci 2 (2):176-185
458. Mote MI, Black KR (1981) Action spectrum and threshold sensitivity of entrainment of circadian
running activity in the cockroach Periplaneta americana. Photochemistry and Photobiology
34 (2):257-265
459. Mukunda L, Miazzi F, Kaltofen S, Hansson BS, Wicher D (2014) Calmodulin modulates insect
odorant receptor function. Cell Calcium 55 (4):191-199
460. Mukunda Shivalingaiah L (2014) Function and regulation of insect olfactory receptors. PhD
thesis, Friedrich-Schiller-University, Jena
461. Mundell SJ, Benovic JL (2000) Selective regulation of endogenous G protein-coupled receptors
by arrestins in HEK293 cells. J Biol Chem 275 (17):12900-12908
462. Murad A, Emery-Le M, Emery P (2007) A subset of dorsal neurons modulates circadian behavior
and light responses in Drosophila. Neuron 53 (5):689-701
463. Muraro NI, Colque CC, Ceriani MF The firing mode of clock neurons in Drosophila is established
by both intrinsic and synaptic factors. In: XII Latin American Symposium on Chronobiology,
Mendoza, Argentina, 2013a.
464. Muraro NI, Pirez N, Ceriani MF (2013b) The circadian system: plasticity at many levels.
Neuroscience 247:280-293
465. Myers EM, Yu J, Sehgal A (2003) Circadian control of eclosion: interaction between a central and
peripheral clock in Drosophila melanogaster. Curr Biol 13 (6):526-533
466. Myers MP, Wager-Smith K, Rothenfluh-Hilfiker A, Young MW (1996) Light-induced degradation
of TIMELESS and entrainment of the Drosophila circadian clock. Science 271 (5256):17361740
262
Bibliography
467. Nadasdy Z (2010) Binding by asynchrony: the neuronal phase code. Front Neurosci 4:51
468. Naidoo N, Song W, Hunter-Ensor M, Sehgal A (1999) A role for the proteasome in the light
response of the timeless clock protein. Science 285 (5434):1737-1741
469. Nakagawa T, Vosshall LB (2009) Controversy and consensus: noncanonical signaling mechanisms
in the insect olfactory system. Curr Opin Neurobiol 19 (3):284-292
470. Nakagawa T, Touhara K (2013) Extracellular modulation of the silkmoth sex pheromone receptor
activity by cyclic nucleotides. PLoS One 8 (6):e63774
471. Nakagawa T, Sakurai T, Nishioka T, Touhara K (2005) Insect sex-pheromone signals mediated by
specific combinations of olfactory receptors. Science 307 (5715):1638-1642
472. Nakagawa T, Pellegrino M, Sato K, Vosshall LB, Touhara K (2012) Amino Acid residues
contributing to function of the heteromeric insect olfactory receptor complex. PLoS One 7
(3):e32372
473. Narahashi T, Zhao X, Ikeda T, Nagata K, Yeh JZ (2007) Differential actions of insecticides on
target sites: basis for selective toxicity. Hum Exp Toxicol 26 (4):361-366
474. Narahashi T, Zhao X, Ikeda T, Salgado VL, Yeh JZ (2010) Glutamate-activated chloride channels:
Unique fipronil targets present in insects but not in mammals. Pestic Biochem Physiol 97
(2):149-152
475. Nässel DR, Persson MG, Muren JE (2000) Baratin, a nonamidated neurostimulating
neuropeptide, isolated from cockroach brain: distribution and actions in the cockroach and
locust nervous systems. J Comp Neurol 422 (2):267-286
476. Nässel DR, Shiga S, Wikstrand EM, Rao KR (1991) Pigment-dispersing hormone-immunoreactive
neurons and their relation to serotonergic neurons in the blowfly and cockroach visual
system. Cell Tissue Res 266 (3):511-523
477. Neuhaus EM, Gisselmann G, Zhang W, Dooley R, Stortkuhl K, Hatt H (2005) Odorant receptor
heterodimerization in the olfactory system of Drosophila melanogaster. Nat Neurosci 8
(1):15-17
478. Newland CF, Cull-Candy SG (1992) On the mechanism of action of picrotoxin on GABA receptor
channels in dissociated sympathetic neurones of the rat. J Physiol 447:191-213
479. Ng FS, Tangredi MM, Jackson FR (2011) Glial cells physiologically modulate clock neurons and
circadian behavior in a calcium-dependent manner. Curr Biol 21 (8):625-634
480. Nichols AS, Chen S, Luetje CW (2011) Subunit contributions to insect olfactory receptor function:
channel block and odorant recognition. Chem Senses 36 (9):781-790
481. Nishiitsutsuji-Uwo J, Pittendrigh CS (1968a) Central nervous system control of circadian
rhythmicity in the cockroach. II. The pathway of light signals that entrain the rhythm. Z Vergl
Physiol 58 (1):1-13
482. Nishiitsutsuji-Uwo J, Pittendrigh C (1968b) Central nervous system control of circadian
rhythmicity in the cockroach. III. The optic lobes, locus of the driving oscillation ? Z Vergl
Physiol 58 (1):14-46
483. Nitabach MN, Blau J, Holmes TC (2002) Electrical silencing of Drosophila pacemaker neurons
stops the free-running circadian clock. Cell 109 (4):485-495
484. Nitabach MN, Sheeba V, Vera DA, Blau J, Holmes TC (2005) Membrane electrical excitability is
necessary for the free-running larval Drosophila circadian clock. J Neurobiol 62 (1):1-13
485. Nitabach MN, Wu Y, Sheeba V, Lemon WC, Strumbos J, Zelensky PK, White BH, Holmes TC
(2006) Electrical hyperexcitation of lateral ventral pacemaker neurons desynchronizes
downstream circadian oscillators in the fly circadian circuit and induces multiple behavioral
periods. J Neurosci 26 (2):479-489
486. Nolte A, Funk NW, Mukunda L, Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M
(2013) In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of
pheromone-transduction in Manduca sexta. PLoS One 8 (5):e62648
487. Numann R, Catterall WA, Scheuer T (1991) Functional modulation of brain sodium channels by
protein kinase C phosphorylation. Science 254 (5028):115-118
263
Bibliography
488. Numberger M, Draguhn A (1996) Patch-Clamp-Technik. Spektrum Akademischer Verlag GmbH,
Heidelberg, Berlin, Oxoford
489. O'Dowd DK (1995) Voltage-gated currents and firing properties of embryonic Drosophila
neurons grown in a chemically defined medium. J Neurobiol 27 (1):113-126
490. Offermanns S, Simon MI (1995) Gα15 and Gα16 couple a wide variety of receptors to
phospholipase C. J Biol Chem 270 (25):15175-15180
491. Olafson PU (2013) Molecular characterization and immunolocalization of the olfactory coreceptor Orco from two blood-feeding muscid flies, the stable fly (Stomoxys calcitrans, L.)
and the horn fly (Haematobia irritans irritans, L.). Insect Mol Biol 22 (2):131-142
492. Olsson SB, Getahun MN, Wicher D, Hansson BS (2011) Piezo controlled microinjection: an in vivo
complement for in vitro sensory studies in insects. J Neurosci Methods 201 (2):385-389
493. Ono K, Fozzard HA (1992) Phosphorylation restores activity of L-type calcium channels after
rundown in inside-out patches from rabbit cardiac cells. J Physiol 454:673-688
494. Ordway RW, Singer JJ, Walsh Jr JV (1991) Direct regulation of ion channels by fatty acids. Trends
in Neurosciences 14 (3):96-100
495. Osterrieder W, Holck M (1989) In vitro pharmacologic profile of Ro 40-5967, a novel Ca2+
channel blocker with potent vasodilator but weak inotropic action. J Cardiovasc Pharmacol
13 (5):754-759
496. Ouyang Y, Andersson CR, Kondo T, Golden SS, Johnson CH (1998) Resonating circadian clocks
enhance fitness in cyanobacteria. Proc Natl Acad Sci U S A 95 (15):8660-8664
497. Page T (1978) Interactions between bilaterally paired components of the cockroach circadian
system. J Comp Physiol 124 (3):225-236
498. Page T (1983a) Effects of optic-tract regeneration on internal coupling in the circadian system of
the cockroach. J Comp Physiol 153 (3):353-363
499. Page T (1983b) Regeneration of the optic tracts and circadian pacemaker activity in the
cockroach Leucophaea maderae. J Comp Physiol 152 (2):231-240
500. Page TL (1981) Effects of localized low-temperature pulses on the cockroach circadian
pacemaker. Am J Physiol 240 (3):R144-150
501. Page TL (1982) Transplantation of the cockroach circadian pacemaker. Science 216 (4541):73-75
502. Page TL (1987) Serotonin phase-shifts the circadian rhythm of locomotor activity in the
cockroach. J Biol Rhythms 2 (1):23-34
503. Page TL (1990) Circadian organization in the cockroach. In: Huber I, Masler EP, Rao BR (eds)
Cockroaches as models for neurobiology: Applications in biomedical research (Volume II), vol
2. CRC Press, Boca Raton, FL, pp 225-245
504. Page TL, Barrett RK (1989) Effects of light on circadian pacemaker development. II. Responses to
light. J Comp Physiol A 165 (1):51-59
505. Page TL, Koelling E (2003) Circadian rhythm in olfactory response in the antennae controlled by
the optic lobe in the cockroach. J Insect Physiol 49 (7):697-707
506. Page TL, Caldarola PC, Pittendrigh CS (1977) Mutual entrainment of bilaterally distributed
circadian pacemaker. Proc Natl Acad Sci U S A 74 (3):1277-1281
507. Pakhotin P, Harmar AJ, Verkhratsky A, Piggins H (2006) VIP receptors control excitability of
suprachiasmatic nuclei neurons. Pflugers Arch 452 (1):7-15
508. Parisky KM, Agosto J, Pulver SR, Shang Y, Kuklin E, Hodge JJ, Kang K, Liu X, Garrity PA, Rosbash
M, Griffith LC (2008) PDF cells are a GABA-responsive wake-promoting component of the
Drosophila sleep circuit. Neuron 60 (4):672-682
509. Park D, Griffith LC (2006) Electrophysiological and anatomical characterization of PDF-positive
clock neurons in the intact adult Drosophila brain. J Neurophysiol 95 (6):3955-3960
510. Park JH, Hall JC (1998) Isolation and chronobiological analysis of a neuropeptide pigmentdispersing factor gene in Drosophila melanogaster. J Biol Rhythms 13 (3):219-228
511. Park JH, Helfrich-Förster C, Lee G, Liu L, Rosbash M, Hall JC (2000) Differential regulation of
circadian pacemaker output by separate clock genes in Drosophila. Proc Natl Acad Sci U S A
97 (7):3608-3613
264
Bibliography
512. Parmentier ML, Pin JP, Bockaert J, Grau Y (1996) Cloning and functional expression of a
Drosophila metabotropic glutamate receptor expressed in the embryonic CNS. J Neurosci 16
(21):6687-6694
513. Pask GM, Jones PL, Rutzler M, Rinker DC, Zwiebel LJ (2011) Heteromeric anopheline odorant
receptors exhibit distinct channel properties. PLoS One 6 (12):e28774
514. Pask GM, Bobkov YV, Corey EA, Ache BW, Zwiebel LJ (2013) Blockade of insect odorant receptor
currents by amiloride derivatives. Chem Senses 38 (3):221-229
515. Patch HM, Velarde RA, Walden KK, Robertson HM (2009) A candidate pheromone receptor and
two odorant receptors of the hawkmoth Manduca sexta. Chem Senses 34 (4):305-316
516. Payne TL, Shorey HH, Gaston LK (1970) Sex pheromones of noctuid moths: factors influencing
antennal responsiveness in males of Trichoplusia ni. J Insect Physiol 16 (6):1043-1055
517. Pellegrino M, Steinbach N, Stensmyr MC, Hansson BS, Vosshall LB (2011) A natural
polymorphism alters odour and DEET sensitivity in an insect odorant receptor. Nature 478
(7370):511-514
518. Peñalva-Arana DC, Lynch M, Robertson HM (2009) The chemoreceptor genes of the waterflea
Daphnia pulex: many Grs but no Ors. BMC Evol Biol 9:79
519. Peng Y, Stoleru D, Levine JD, Hall JC, Rosbash M (2003) Drosophila free-running rhythms require
intercellular communication. PLoS Biol 1 (1):E13
520. Penzlin H (2005) Lehrbuch der Tierphysiologie. 7th edn. Spektrum Akademischer Verlag,
Heidelberg
521. Peron S, Gabbiani F (2009) Spike frequency adaptation mediates looming stimulus selectivity in
a collision-detecting neuron. Nat Neurosci 12 (3):318-326
522. Persson MG, Eklund MB, Dircksen H, Muren JE, Nassel DR (2001) Pigment-dispersing factor in
the locust abdominal ganglia may have roles as circulating neurohormone and central
neuromodulator. J Neurobiol 48 (1):19-41
523. Peschel N, Veleri S, Stanewsky R (2006) Veela defines a molecular link between Cryptochrome
and Timeless in the light-input pathway to Drosophila's circadian clock. Proc Natl Acad Sci U S
A 103 (46):17313-17318
524. Peschel N, Chen KF, Szabo G, Stanewsky R (2009) Light-dependent interactions between the
Drosophila circadian clock factors Cryptochrome, Jetlag, and Timeless. Curr Biol 19 (3):241247
525. Peters Jr T (1985) Serum albumin. In: C.B. Anfinsen JTE, Frederic MR (eds) Advances in Protein
Chemistry, vol Volume 37. Academic Press, pp 161-245
526. Peterson BZ, DeMaria CD, Adelman JP, Yue DT (1999) Calmodulin is the Ca2+ sensor for Ca2+ dependent inactivation of L-type calcium channels. Neuron 22 (3):549-558
527. Petri B, Stengl M (1997) Pigment-dispersing hormone shifts the phase of the circadian
pacemaker of the cockroach Leucophaea maderae. J Neurosci 17 (11):4087-4093
528. Petri B, Stengl M (1999) Presumptive insect circadian pacemakers in vitro: immunocytochemical
characterization of cultured pigment-dispersing hormone-immunoreactive neurons of
Leucophaea maderae. Cell Tissue Res 296 (3):635-643
529. Petri B, Stengl M (2001) Phase response curves of a molecular model oscillator: implications for
mutual coupling of paired oscillators. J Biol Rhythms 16 (2):125-141
530. Petri B, Stengl M, Wurden S, Homberg U (1995) Immunocytochemical characterization of the
accessory medulla in the cockroach Leucophaea maderae. Cell Tissue Res 282 (1):3-19
531. Petri B, Homberg U, Loesel R, Stengl M (2002) Evidence for a role of GABA and Mas-allatotropin
in photic entrainment of the circadian clock of the cockroach Leucophaea maderae. J Exp Biol
205 (Pt 10):1459-1469
532. Picot M, Cusumano P, Klarsfeld A, Ueda R, Rouyer F (2007) Light activates output from evening
neurons and inhibits output from morning neurons in the Drosophila circadian clock. PLoS
Biol 5 (11):e315
533. Pikovsky A, Rosenblum M, Kurths J (2001) Synchronization. A universal concept in nonlinear
sciences. Cambrigde University Press, Cambridge
265
Bibliography
534. Pírez N, Christmann BL, Griffith LC (2013) Daily rhythms in locomotor circuits in Drosophila
involve PDF. J Neurophysiol 110 (3):700-708
535. Pittendrigh CS (1993) Temporal organization: reflections of a Darwinian clock-watcher. Annu
Rev Physiol 55:16-54
536. Pittendrigh CS, Bruce V (1957) An oscillator model for biological clocks. In: Rudnik D (ed)
Rhythmic and synthetic processes in growth. Princeton University Press, Princeton, NJ, p 75
537. Pittendrigh CS, Caldarola PC (1973) General homeostasis of the frequency of circadian
oscillations. Proc Natl Acad Sci U S A 70 (9):2697-2701
538. Pitts RJ, Fox AN, Zwiebel LJ (2004) A highly conserved candidate chemoreceptor expressed in
both olfactory and gustatory tissues in the malaria vector Anopheles gambiae. Proc Natl Acad
Sci U S A 101 (14):5058-5063
539. Pitts RJ, Liu C, Zhou X, Malpartida JC, Zwiebel LJ (2014) Odorant receptor-mediated sperm
activation in disease vector mosquitoes. Proc Natl Acad Sci U S A 111 (7):2566-2571
540. Plautz JD, Kaneko M, Hall JC, Kay SA (1997) Independent photoreceptive circadian clocks
throughout Drosophila. Science 278 (5343):1632-1635
541. Pophof B, Van der Goes van Naters W (2002) Activation and inhibition of the transduction
process in silkmoth olfactory receptor neurons. Chem Senses 27 (5):435-443
542. Pregitzer P, Greschista M, Breer H, Krieger J (2014) The sensory neurone membrane protein
SNMP1 contributes to the sensitivity of a pheromone detection system. Insect Mol Biol
543. Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW (1998) double-time is a novel
Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94 (1):83-95
544. Prosser RA, Gillette MU (1989) The mammalian circadian clock in the suprachiasmatic nuclei is
reset in vitro by cAMP. J Neurosci 9 (3):1073-1081
545. Prosser RA, McArthur AJ, Gillette MU (1989) cGMP induces phase shifts of a mammalian
circadian pacemaker at night, in antiphase to cAMP effects. Proc Natl Acad Sci U S A 86
(17):6812-6815
546. Prut Y, Perlmutter SI (2003) Firing properties of spinal interneurons during voluntary movement.
I. State-dependent regularity of firing. J Neurosci 23 (29):9600-9610
547. Qin N, Olcese R, Bransby M, Lin T, Birnbaumer L (1999) Ca2+-induced inhibition of the cardiac
Ca2+ channel depends on calmodulin. Proc Natl Acad Sci U S A 96 (5):2435-2438
548. Rae J, Cooper K, Gates P, Watsky M (1991) Low access resistance perforated patch recordings
using amphotericin B. J Neurosci Methods 37 (1):15-26
549. Ramirez JM, Tryba AK, Pena F (2004) Pacemaker neurons and neuronal networks: an integrative
view. Curr Opin Neurobiol 14 (6):665-674
550. Rao KR, Riehm JP (1988) Pigment-dispersing hormones: a novel family of neuropeptides from
arthropods. Peptides 9 Suppl 1:153-159
551. Rao KR, Riehm JP (1989) The Pigment-Dispersing Hormone Family: Chemistry, Structure-Activity
Relations, and Distribution. The Biological Bulletin 177 (2):225-229
552. Rao KR, Mohrherr CJ, Riehm JP, Zahnow CA, Norton S, Johnson L, Tarr GE (1987) Primary
structure of an analog of crustacean pigment-dispersing hormone from the lubber
grasshopper Romalea microptera. J Biol Chem 262 (6):2672-2675
553. Ray K, Ganguly R (1992) The Drosophila G protein  subunit gene (D-G1) produces three
developmentally regulated transcripts and is predominantly expressed in the central nervous
system. J Biol Chem 267 (9):6086-6092
554. Raymond V, Sattelle DB (2002) Novel animal-health drug targets from ligand-gated chloride
channels. Nat Rev Drug Discov 1 (6):427-436
555. Raymond V, Sattelle DB, Lapied B (2000) Co-existence in DUM neurones of two GluCl channels
that differ in their picrotoxin sensitivity. Neuroreport 11 (12):2695-2701
556. Rea MA (1990) VIP-stimulated cyclic AMP accumulation in the suprachiasmatic hypothalamus.
Brain Res Bull 25 (6):843-847
557. Reischig T (2003) Identification and Characterisation of the Circadian Pacemaker of the
Cockroach Leucophaea maderae. PhD Thesis, Philipps-University, Marburg
266
Bibliography
558.
Reischig T, Stengl M (1996) Morphology and pigment-dispersing hormone
immunocytochemistry of the accessory medulla, the presumptive circadian pacemaker of the
cockroach Leucophaea maderae: A light- and electron-microscopic study. Cell and Tissue
Research 285 (2):305-319
559. Reischig T, Stengl M (2002) Optic lobe commissures in a three-dimensional brain model of the
cockroach Leucophaea maderae: A search for the circadian coupling pathways. Journal of
Comparative Neurology 443 (4):388-400
560. Reischig T, Stengl M (2003a) Ectopic transplantation of the accessory medulla restores circadian
locomotor rhythms in arrhythmic cockroaches (Leucophaea maderae). J Exp Biol 206 (Pt
11):1877-1886
561. Reischig T, Stengl M (2003b) Ultrastructure of pigment-dispersing hormone-immunoreactive
neurons in a three-dimensional model of the accessory medulla of the cockroach Leucophaea
maderae. Cell and Tissue Research 314 (3):421-435
562. Reischig T, Petri B, Stengl M (2004) Pigment-dispersing hormone (PDH)-immunoreactive
neurons form a direct coupling pathway between the bilaterally symmetric circadian
pacemakers of the cockroach Leucophaea maderae. Cell and Tissue Research 318 (3):553-564
563. Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH (1999) A pdf neuropeptide gene mutation and
ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in
Drosophila. Cell 99 (7):791-802
564. Reppert SM, Tsai T, Roca AL, Sauman I (1994) Cloning of a structural and functional homolog of
the circadian clock gene period from the giant silkmoth Antheraea pernyi. Neuron 13
(5):1167-1176
565. Richier B, Michard-Vanhee C, Lamouroux A, Papin C, Rouyer F (2008) The clockwork orange
Drosophila protein functions as both an activator and a repressor of clock gene expression. J
Biol Rhythms 23 (2):103-116
566. Rieger D, Shafer OT, Tomioka K, Helfrich-Förster C (2006) Functional analysis of circadian
pacemaker neurons in Drosophila melanogaster. J Neurosci 26 (9):2531-2543
567. Riesgo-Escovar J, Raha D, Carlson JR (1995) Requirement for a phospholipase C in odor
response: overlap between olfaction and vision in Drosophila. Proc Natl Acad Sci U S A 92
(7):2864-2868
568. Riesgo-Escovar JR, Woodard C, Carlson JR (1994) Olfactory physiology in the Drosophila
maxillary palp requires the visual system gene rdgB. J Comp Physiol A 175 (6):687-693
569. Riffell JA, Alarcon R, Abrell L, Davidowitz G, Bronstein JL, Hildebrand JG (2008) Behavioral
consequences of innate preferences and olfactory learning in hawkmoth-flower interactions.
570. Roberts SK (1965) Photoreception and entrainment of cockroach activity rhythms. Science 148
(3672):958-959
571. Roberts SK (1974) Circadian Rhythms in cockroaches - Effects of optic lobe lesions. J Comp
Physiol 88 (1):21-30
572. Robertson HM, Gadau J, Wanner KW (2010) The insect chemoreceptor superfamily of the
parasitoid jewel wasp Nasonia vitripennis. Insect Mol Biol 19 Suppl 1:121-136
573. Rogers ME, Krieger J, Vogt RG (2001a) Antennal SNMPs (sensory neuron membrane proteins) of
Lepidoptera define a unique family of invertebrate CD36-like proteins. J Neurobiol 49 (1):4761
574. Rogers ME, Steinbrecht RA, Vogt RG (2001b) Expression of SNMP-1 in olfactory neurons and
sensilla of male and female antennae of the silkmoth Antheraea polyphemus. Cell Tissue Res
303 (3):433-446
575. Rogers ME, Sun M, Lerner MR, Vogt RG (1997) SNMP-1, a novel membrane protein of olfactory
neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of
membrane proteins. J Biol Chem 272 (23):14792-14799
576. Röllecke K, Werner M, Ziemba PM, Neuhaus EM, Hatt H, Gisselmann G (2013) Amiloride
derivatives are effective blockers of insect odorant receptors. Chem Senses 38 (3):231-236
267
Bibliography
577. Romanin C, Grösswagen P, Schindler H (1991) Calpastatin and nucleotides stabilize cardiac
calcium channel activity in excised patches. Pflugers Arch 418 (1-2):86-92
578. Rosato E, Codd V, Mazzotta G, Piccin A, Zordan M, Costa R, Kyriacou CP (2001) Light-dependent
interaction between Drosophila CRY and the clock protein PER mediated by the carboxy
terminus of CRY. Curr Biol 11 (12):909-917
579. Rosen W (2002) Endogenous control of circadian rhythms of pheromone production in the
turnip moth, Agrotis segetum. Arch Insect Biochem Physiol 50 (1):21-30
580. Rosen WQ, Han GB, Lofstedt C (2003) The circadian rhythm of the sex-pheromone-mediated
behavioral response in the turnip moth, Agrotis segetum, is not controlled at the peripheral
level. J Biol Rhythms 18 (5):402-408
581. Roth RL, Sokolove PG (1975) Histological evidence for direct connections between the optic
lobes of the cockroach Leucophaea maderae. Brain Res 87 (1):23-39
582. Rothenfluh A, Young MW, Saez L (2000) A TIMELESS-independent function for PERIOD proteins
in the Drosophila clock. Neuron 26 (2):505-514
583. Ruben M, Drapeau MD, Mizrak D, Blau J (2012) A mechanism for circadian control of pacemaker
neuron excitability. J Biol Rhythms 27 (5):353-364
584. Rumbo ER, Kaissling K-E (1989) Temporal resolution of odour pulses by three types of
pheromone receptor cells in Antheraea polyphemus. J Comp Physiol A 165:281-291
585. Rutila JE, Suri V, Le M, So WV, Rosbash M, Hall JC (1998) CYCLE is a second bHLH-PAS clock
protein essential for circadian rhythmicity and transcription of Drosophila period and
timeless. Cell 93 (5):805-814
586. Rützler M, Lu T, Zwiebel LJ (2006) Gα encoding gene family of the malaria vector mosquito
Anopheles gambiae: expression analysis and immunolocalization of a Gαq and a Gαo in female
antennae. J Comp Neurol 499 (4):533-545
587. Rymer J, Bauernfeind AL, Brown S, Page TL (2007) Circadian rhythms in the mating behavior of
the cockroach, Leucophaea maderae. J Biol Rhythms 22 (1):43-57
588. Rytz R, Croset V, Benton R (2013) Ionotropic receptors (IRs): chemosensory ionotropic
glutamate receptors in Drosophila and beyond. Insect Biochem Mol Biol 43 (9):888-897
589. Saez L, Young MW (1996) Regulation of nuclear entry of the Drosophila clock proteins Period
and Timeless. Neuron 17 (5):911-920
590. Saez L, Derasmo M, Meyer P, Stieglitz J, Young MW (2011) A key temporal delay in the circadian
cycle of Drosophila is mediated by a nuclear localization signal in the Timeless protein.
Genetics 188 (3):591-600
591. Saifullah AS, Page TL (2009) Circadian regulation of olfactory receptor neurons in the cockroach
antenna. J Biol Rhythms 24 (2):144-152
592. Sakai T, Ishida N (2001) Circadian rhythms of female mating activity governed by clock genes in
Drosophila. Proc Natl Acad Sci U S A 98 (16):9221-9225
593. Sakurai T, Nakagawa T, Mitsuno H, Mori H, Endo Y, Tanoue S, Yasukochi Y, Touhara K, Nishioka T
(2004) Identification and functional characterization of a sex pheromone receptor in the
silkmoth Bombyx mori. Proc Natl Acad Sci U S A 101 (47):16653-16658
594. Sandrelli F, Costa R, Kyriacou CP, Rosato E (2008) Comparative analysis of circadian clock genes
in insects. Insect Mol Biol 17 (5):447-463
595. Sane SP (2006) Induced airflow in flying insects I. A theoretical model of the induced flow. J Exp
Biol 209 (1):32-42
596. Sane SP, Jacobson NP (2006) Induced airflow in flying insects II. Measurement of induced flow. J
Exp Biol 209 (1):43-56
597. Sargsyan V, Getahun MN, Llanos SL, Olsson SB, Hansson BS, Wicher D (2011) Phosphorylation
via PKC regulates the function of the Drosophila odorant co-receptor. Front Cell Neurosci 5:5
598. Sasaki M, Riddiford LM (1984) Regulation of reproductive behaviour and egg maturation in the
tobacco hawk moth, Manduca sexta. Physiological Entomology 9 (3):315-327
599. Sathyanarayanan S, Zheng X, Xiao R, Sehgal A (2004) Posttranslational regulation of Drosophila
PERIOD protein by protein phosphatase 2A. Cell 116 (4):603-615
268
Bibliography
600. Sato K, Pellegrino M, Nakagawa T, Vosshall LB, Touhara K (2008) Insect olfactory receptors are
heteromeric ligand-gated ion channels. Nature 452 (7190):1002-1006
601. Sato S, Chuman Y, Matsushima A, Tominaga Y, Shimohigashi Y, Shimohigashi M (2002) A
circadian neuropeptide, pigment-dispersing factor-PDF, in the last-summer cicada Meimuna
opalifera: cDNA cloning and immunocytochemistry. Zoolog Sci 19 (8):821-828
602. Sauman I, Reppert SM (1996) Circadian clock neurons in the silkmoth Antheraea pernyi: novel
mechanisms of Period protein regulation. Neuron 17 (5):889-900
603. Schaap J, Pennartz CM, Meijer JH (2003) Electrophysiology of the circadian pacemaker in
mammals. Chronobiol Int 20 (2):171-188
604. Schäfer S, Rosenboom H, Menzel R (1994) Ionic currents of Kenyon cells from the mushroom
body of the honeybee. J Neurosci 14 (8):4600-4612
605. Schendzielorz J (2013) Analysis of the circadian system of the cockroach Rhyparobia
(Leucophaea) maderae. PhD Thesis, University of Kassel, Kassel
606. Schendzielorz J, Stengl M (2014) Candidates for the light entrainment pathway to the circadian
clock of the Madeira cockroach Rhyparobia maderae. Cell Tissue Res 355 (2):447-462
607. Schendzielorz J, Schendzielorz T, Arendt A, Stengl M (2014) Bimodal oscillations of cyclic
nucleotide concentrations in the circadian system of the Madeira cockroach Rhyparobia
maderae. J Biol Rhythms
608. Schendzielorz T, Peters W, Boekhoff I, Stengl M (2012) Time of day changes in cyclic nucleotides
are modified via octopamine and pheromone in antennae of the Madeira cockroach. J Biol
Rhythms 27 (5):388-397
609. Schendzielorz T, Schirmer K, Stolte P, Stengl M (2015) Octopamine regulates antennal sensory
neurons via daytime-dependent changes in cAMP and IP3 levels in the hawkmoth Manduca
sexta. PLoS One 10 (3):e0121230
610. Schleicher S, Boekhoff I, Konietzko U, Breer H (1994) Pheromone-induced phosphorylation of
antennal proteins from insects. J Comp Physiol B 164 (1):76-80
611. Schmidt CJ, Garen-Fazio S, Chow YK, Neer EJ (1989) Neuronal expression of a newly identified
Drosophila melanogaster G protein alpha O subunit. Cell Regul 1 (1):125-134
612. Schmitt H, Meves H (1994) Bovine serum albumin selectively increases the low-voltageactivated calcium current of NG108-15 neuroblastoma x glioma cells. Brain Res 656 (2):375380
613. Schneider D (1969) Insect olfaction: deciphering system for chemical messages. Science 163
(3871):1031-1037
614. Schneider D, Lacher V, Kaissling K-E (1964) Die Reaktionsweise und das Reaktionsspektrum von
Riechzellen bei Antheraea pernyi (Lepidoptera, Saturniidae). Z Vergl Physiol 48 (6):632-662
615. Schneider NL, Stengl M (2005) Pigment-dispersing factor and GABA synchronize cells of the
isolated circadian clock of the cockroach Leucophaea maderae. J Neurosci 25 (21):5138-5147
616. Schneider NL, Stengl M (2006) Gap junctions between accessory medulla neurons appear to
synchronize circadian clock cells of the cockroach Leucophaea maderae. J Neurophysiol 95
(3):1996-2002
617. Schneider NL, Stengl M (2007) Extracellular long-term recordings of the isolated accessory
medulla, the circadian pacemaker center of the cockroach Leucophaea maderae, reveal
ultradian and hint circadian rhythms. J Comp Physiol A Neuroethol Sens Neural Behav Physiol
193 (1):35-42
618. Schuckel J, Siwicki KK, Stengl M (2007) Putative circadian pacemaker cells in the antenna of the
hawkmoth Manduca sexta. Cell Tissue Res 330 (2):271-278
619. Schulz S, Huber A, Schwab K, Paulsen R (1999) A novel G isolated from Drosophila constitutes a
visual G protein gamma subunit of the fly compound eye. J Biol Chem 274 (53):37605-37610
620. Schulze J, Schendzielorz T, Neupert S, Predel R, Stengl M (2013) Neuropeptidergic input
pathways to the circadian pacemaker center of the Madeira cockroach analysed with an
improved injection technique. Eur J Neurosci
269
Bibliography
621. Schulze J, Neupert S, Schmidt L, Predel R, Lamkemeyer T, Homberg U, Stengl M (2012)
Myoinhibitory peptides in the brain of the cockroach Leucophaea maderae and colocalization
with pigment-dispersing factor in circadian pacemaker cells. J Comp Neurol 520 (5):10781097
622. Sedelnikova A, Erkkila BE, Harris H, Zakharkin SO, Weiss DS (2006) Stoichiometry of a pore
mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor. J Physiol
577 (Pt 2):569-577
623. Sehgal A, Price JL, Man B, Young MW (1994) Loss of circadian behavioral rhythms and per RNA
oscillations in the Drosophila mutant timeless. Science 263 (5153):1603-1606
624. Sehgal A, Rothenfluh-Hilfiker A, Hunter-Ensor M, Chen Y, Myers MP, Young MW (1995)
Rhythmic expression of timeless: a basis for promoting circadian cycles in period gene
autoregulation. Science 270 (5237):808-810
625. Seluzicki A, Flourakis M, Kula-Eversole E, Zhang L, Kilman V, Allada R (2014) Dual PDF signaling
pathways reset clocks via TIMELESS and acutely excite target neurons to control circadian
behavior. PLoS Biol 12 (3):e1001810
626. Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, Yoshihara Y, Sakano H (2003) Negative
feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science
302 (5653):2088-2094
627. Seydl K, Karlsson J-O, Dominik A, Gruber H, Romanin C (1995) Action of calpastatin in prevention
of cardiac L-type Ca2+ channel run-down cannot be mimicked by synthetic calpain inhibitors.
Pflugers Arch 429 (4):503-510
628. Shafer OT, Taghert PH (2009) RNA-interference knockdown of Drosophila pigment dispersing
factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions. PLoS
One 4 (12):e8298
629. Shafer OT, Rosbash M, Truman JW (2002) Sequential nuclear accumulation of the clock proteins
period and timeless in the pacemaker neurons of Drosophila melanogaster. J Neurosci 22
(14):5946-5954
630. Shafer OT, Helfrich-Förster C, Renn SC, Taghert PH (2006) Reevaluation of Drosophila
melanogaster's neuronal circadian pacemakers reveals new neuronal classes. J Comp Neurol
498 (2):180-193
631. Shafer OT, Kim DJ, Dunbar-Yaffe R, Nikolaev VO, Lohse MJ, Taghert PH (2008) Widespread
receptivity to neuropeptide PDF throughout the neuronal circadian clock network of
Drosophila revealed by real-time cyclic AMP imaging. Neuron 58 (2):223-237
632. Shanbhag SR, Muller B, Steinbrecht RA (1999) Atlas of olfactory organs of Drosophila
melanogaster - 1. Types, external organization, innervation and distribution of olfactory
sensilla. International Journal of Insect Morphology & Embryology 28 (4):377-397
633. Shang Y, Griffith LC, Rosbash M (2008) Light-arousal and circadian photoreception circuits
intersect at the large PDF cells of the Drosophila brain. Proc Natl Acad Sci U S A 105
(50):19587-19594
634. Shang Y, Haynes P, Pirez N, Harrington KI, Guo F, Pollack J, Hong P, Griffith LC, Rosbash M (2011)
Imaging analysis of clock neurons reveals light buffers the wake-promoting effect of
dopamine. Nat Neurosci 14 (7):889-895
635. Sheeba V, Fogle KJ, Holmes TC (2010) Persistence of morning anticipation behavior and high
amplitude morning startle response following functional loss of small ventral lateral neurons
in Drosophila. PLoS One 5 (7):e11628
636. Sheeba V, Gu H, Sharma VK, O'Dowd DK, Holmes TC (2008a) Circadian- and light-dependent
regulation of resting membrane potential and spontaneous action potential firing of
Drosophila circadian pacemaker neurons. J Neurophysiol 99 (2):976-988
637. Sheeba V, Sharma VK, Gu H, Chou YT, O'Dowd DK, Holmes TC (2008b) Pigment dispersing factordependent and -independent circadian locomotor behavioral rhythms. J Neurosci 28 (1):217227
270
Bibliography
638. Sheeba V, Fogle KJ, Kaneko M, Rashid S, Chou YT, Sharma VK, Holmes TC (2008c) Large ventral
lateral neurons modulate arousal and sleep in Drosophila. Curr Biol 18 (20):1537-1545
639. Sheward WJ, Lutz EM, Harmar AJ (1995) The distribution of vasoactive intestinal peptide2
receptor messenger RNA in the rat brain and pituitary gland as assessed by in situ
hybridization. Neuroscience 67 (2):409-418
640. Shinohara K, Funabashi T, Kimura F (1999) Temporal profiles of vasoactive intestinal polypeptide
precursor mRNA and its receptor mRNA in the rat suprachiasmatic nucleus. Brain Res Mol
Brain Res 63 (2):262-267
641. Shinohara K, Funabashi T, Mitushima D, Kimura F (2000a) Effects of gap junction blocker on
vasopressin and vasoactive intestinal polypeptide rhythms in the rat suprachiasmatic nucleus
in vitro. Neurosci Res 38 (1):43-47
642. Shinohara K, Hiruma H, Funabashi T, Kimura F (2000b) GABAergic modulation of gap junction
communication in slice cultures of the rat suprachiasmatic nucleus. Neuroscience 96 (3):591596
643. Silbering AF, Benton R (2010) Ionotropic and metabotropic mechanisms in chemoreception:
'chance or design'? EMBO Rep 11 (3):173-179
644. Silvegren G, Lofstedt C, Qi Rosen W (2005) Circadian mating activity and effect of pheromone
pre-exposure on pheromone response rhythms in the moth Spodoptera littoralis. J Insect
Physiol 51 (3):277-286
645. Singaravel M, Fujisawa Y, Hisada M, Saifullah AS, Tomioka K (2003) Phase shifts of the circadian
locomotor rhythm induced by pigment-dispersing factor in the cricket Gryllus bimaculatus.
Zoolog Sci 20 (11):1347-1354
646. Singer W, Gray CM (1995) Visual feature integration and the temporal correlation hypothesis.
Annu Rev Neurosci 18:555-586
647. Siwicki KK, Eastman C, Petersen G, Rosbash M, Hall JC (1988) Antibodies to the period gene
product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual
system. Neuron 1 (2):141-150
648. Smadja C, Shi P, Butlin RK, Robertson HM (2009) Large gene family expansions and adaptive
evolution for odorant and gustatory receptors in the pea aphid, Acyrthosiphon pisum. Mol
Biol Evol 26 (9):2073-2086
649. Smart R, Kiely A, Beale M, Vargas E, Carraher C, Kralicek AV, Christie DL, Chen C, Newcomb RD,
Warr CG (2008) Drosophila odorant receptors are novel seven transmembrane domain
proteins that can signal independently of heterotrimeric G proteins. Insect Biochem Mol Biol
38 (8):770-780
650. Soehler S, Stengl M, Reischig T (2011) Circadian pacemaker coupling by multi-peptidergic
neurons in the cockroach Leucophaea maderae. Cell Tissue Res 343 (3):559-577
651. Soehler S, Neupert S, Predel R, Stengl M (2008) Examination of the role of FMRFamide-related
peptides in the circadian clock of the cockroach Leucophaea maderae. Cell Tissue Res 332
(2):257-269
652. Soehler S, Neupert S, Predel R, Nichols R, Stengl M (2007) Localization of leucomyosuppressin in
the brain and circadian clock of the cockroach Leucophaea maderae. Cell Tissue Res 328
(2):443-452
653. Sokolove PG (1975) Localization of the cockroach optic lobe circadian pacemaker with
microlesions. Brain Res 87 (1):13-21
654. Song Y, Cygnar KD, Sagdullaev B, Valley M, Hirsh S, Stephan A, Reisert J, Zhao H (2008) Olfactory
CNG channel desensitization by Ca2+/CaM via the B1b subunit affects response termination
but not sensitivity to recurring stimulation. Neuron 58 (3):374-386
655. Spehr J, Hagendorf S, Weiss J, Spehr M, Leinders-Zufall T, Zufall F (2009) Ca2+ -calmodulin
feedback mediates sensory adaptation and inhibits pheromone-sensitive ion channels in the
vomeronasal organ. J Neurosci 29 (7):2125-2135
656. Srikanth S, Gwack Y (2012) Orai1, STIM1, and their associating partners. J Physiol 590 (Pt
17):4169-4177
271
Bibliography
657. Stanewsky R, Kaneko M, Emery P, Beretta B, Wager-Smith K, Kay SA, Rosbash M, Hall JC (1998)
The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95
(5):681-692
658. Starratt AN, Dahm KH, Allen N, Hildebrand JG, Payne TL, Roller H (1979) Bombykal, a sex
pheromone of the sphinx moth Manduca sexta. Zeitschrift für Naturforschung Section C
Biosciences 34:9-12
659. Stengl M (1993) Intracellular-messenger-mediated cation channels in cultured olfactory
receptor neurons. J Exp Biol 178:125-147
660. Stengl M (1994) Inositol-trisphosphate-dependent calcium currents precede cation currents in
insect olfactory receptor neurons in vitro. J Comp Physiol A Neuroethol Sens Neural Behav
Physiol 174 (2):187-194
661. Stengl M (1995) Pigment-dispersing hormone-immunoreactive fibers persist in crickets which
remain rhythmic after bilateral transection of the optic stalks. Journal of Comparative
Physiology A 176 (2):217-228
662. Stengl M (2010) Pheromone transduction in moths. Front Cell Neurosci 4:133
663. Stengl M, Hildebrand JG (1990) Insect olfactory neurons in vitro: morphological and
immunocytochemical characterization of male-specific antennal receptor cells from
developing antennae of male Manduca sexta. J Neurosci 10 (3):837-847
664. Stengl M, Homberg U (1994) Pigment-dispersing hormone-immunoreactive neurons in the
cockroach Leucophaea maderae share properties with circadian pacemaker neurons. J Comp
Physiol A 175 (2):203-213
665. Stengl M, Funk NW (2013) The role of the coreceptor Orco in insect olfactory transduction. J
Comp Physiol A Neuroethol Sens Neural Behav Physiol 199 (11):897-909
666. Stengl M, Hatt H, Breer H (1992a) Peripheral processes in insect olfaction. Annu Rev Physiol
54:665-681
667. Stengl M, Zufall F, Hatt H, Hildebrand JG (1992b) Olfactory receptor neurons from antennae of
developing male Manduca sexta respond to components of the species-specific sex
pheromone in vitro. J Neurosci 12 (7):2523-2531
668. Stengl M, Zintl R, De Vente J, Nighorn A (2001) Localization of cGMP immunoreactivity and of
soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta. Cell Tissue Res
304 (3):409-421
669. Stieger K (2011) In vivo cAMP-Messungen von circadianen Schrittmacherneuronen der Schabe
Leucophaea maderae - Die cAMP-abhängige Proteinkinase als FRET-Sensor. Diploma thesis,
University of Kassel, Kassel
670. Stocker RF (1994) The organization of the chemosensory system in Drosophila melanogaster: a
review. Cell Tissue Res 275 (1):3-26
671. Stoleru D, Peng Y, Agosto J, Rosbash M (2004) Coupled oscillators control morning and evening
locomotor behaviour of Drosophila. Nature 431 (7010):862-868
672. Stoleru D, Peng Y, Nawathean P, Rosbash M (2005) A resetting signal between Drosophila
pacemakers synchronizes morning and evening activity. Nature 438 (7065):238-242
673. Stoleru D, Nawathean P, Fernandez MP, Menet JS, Ceriani MF, Rosbash M (2007) The Drosophila
circadian network is a seasonal timer. Cell 129 (1):207-219
674. Su CY, Menuz K, Reisert J, Carlson JR (2012) Non-synaptic inhibition between grouped neurons
in an olfactory circuit. Nature 492 (7427):66-71
675. Suh E, Bohbot J, Zwiebel LJ (2014) Peripheral olfactory signaling in insects. Curr Opin Insect Sci
6:86-92
676. Suh J, Jackson FR (2007) Drosophila Ebony activity is required in glia for the circadian regulation
of locomotor activity. Neuron 55 (3):435-447
677. Sun QQ, Prince DA, Huguenard JR (2003) Vasoactive intestinal polypeptide and pituitary
adenylate cyclase-activating polypeptide activate hyperpolarization-activated cationic
current and depolarize thalamocortical neurons in vitro. J Neurosci 23 (7):2751-2758
272
Bibliography
678. Syed Z, Leal WS (2008) Mosquitoes smell and avoid the insect repellent DEET. Proc Natl Acad Sci
U S A 105 (36):13598-13603
679. Szyszka P, Gerkin RC, Galizia CG, Smith BH (2014) High-speed odor transduction and pulse
tracking by insect olfactory receptor neurons. Proc Natl Acad Sci U S A 111 (47):16925-16930
680. Taghert PH, Shafer OT (2006) Mechanisms of clock output in the Drosophila circadian
pacemaker system. J Biol Rhythms 21 (6):445-457
681. Taghert PH, Nitabach MN (2012) Peptide neuromodulation in invertebrate model systems.
Neuron 76 (1):82-97
682. Taghert PH, Hewes RS, Park JH, O'Brien MA, Han M, Peck ME (2001) Multiple amidated
neuropeptides are required for normal circadian locomotor rhythms in Drosophila. J Neurosci
21 (17):6673-6686
683. Talluri S, Bhatt A, Smith DP (1995) Identification of a Drosophila G protein α subunit (dGq α-3)
expressed in chemosensory cells and central neurons. Proc Natl Acad Sci U S A 92 (25):1147511479
684. Talsma AD, Christov CP, Terriente-Felix A, Linneweber GA, Perea D, Wayland M, Shafer OT,
Miguel-Aliaga I (2012) Remote control of renal physiology by the intestinal neuropeptide
pigment-dispersing factor in Drosophila. Proc Natl Acad Sci U S A 109 (30):12177-12182
685. Tanoue S, Krishnan P, Chatterjee A, Hardin PE (2008) G protein-coupled receptor kinase 2 is
required for rhythmic olfactory responses in Drosophila. Curr Biol 18 (11):787-794
686. Tanoue S, Krishnan P, Krishnan B, Dryer SE, Hardin PE (2004) Circadian clocks in antennal
neurons are necessary and sufficient for olfaction rhythms in Drosophila. Curr Biol 14 (8):638649
687. Taylor RW, Romaine IM, Liu C, Murthi P, Jones PL, Waterson AG, Sulikowski GA, Zwiebel LJ
(2012) Structure-activity relationship of a broad-spectrum insect odorant receptor agonist.
ACS Chem Biol 7 (10):1647-1652
688. Tepper JM, Martin LP, Anderson DR (1995) GABAA receptor-mediated inhibition of rat substantia
nigra dopaminergic neurons by pars reticulata projection neurons. J Neurosci 15 (4):30923103
689. Tolbert LP, Hildebrand JG (1981) Organization and synaptic ultrastructure of glomeruli in the
antennal lobes of the moth Manduca sexta: A study using thin sections and freeze-fracture.
Proceedings of the Royal Society of London Series B Biological Sciences 213 (1192):279-301
690. Toma DP, White KP, Hirsch J, Greenspan RJ (2002) Identification of genes involved in Drosophila
melanogaster geotaxis, a complex behavioral trait. Nat Genet 31 (4):349-353
691. Tomioka K, Uryu O, Kamae Y, Umezaki Y, Yoshii T (2012) Peripheral circadian rhythms and their
regulatory mechanism in insects and some other arthropods: a review. J Comp Physiol B 182
(6):729-740
692. Tripathy SJ, Peters OJ, Staudacher EM, Kalwar FR, Hatfield MN, Daly KC (2010) Odors pulsed at
wing beat frequencies are tracked by primary olfactory networks and enhance odor
detection. Front Cell Neurosci 4:1
693. Tritsch GL, Moore GE (1962) Spontaneous decomposition of glutamine in cell culture media. Exp
Cell Res 28:360-364
694. Tsai LT, Bainton RJ, Blau J, Heberlein U (2004) Lmo mutants reveal a novel role for circadian
pacemaker neurons in cocaine-induced behaviors. PLoS Biol 2 (12):e408
695. Tsitoura P, Andronopoulou E, Tsikou D, Agalou A, Papakonstantinou MP, Kotzia GA, Labropoulou
V, Swevers L, Georgoussi Z, Iatrou K (2010) Expression and membrane topology of Anopheles
gambiae odorant receptors in lepidopteran insect cells. PLoS One 5 (11):e15428
696. Tumlinson JH, Brennan MM, Doolittle RE, Mitchell ER, Brabham A, Mazomenos BE, Baumhover
AH, Jackson DM (1989) Identification of a pheromone blend attractive to Manduca sexta (L.)
males in a wind-tunnel. Archives of Insect Biochemistry and Physiology 10 (4):255-271
697. Turner RM, Derryberry SL, Kumar BN, Brittain T, Zwiebel LJ, Newcomb RD, Christie DL (2014)
Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from
273
Bibliography
Drosophila melanogaster reveals differential effects on agonist- and odorant/tuning
receptor-dependent activation. J Biol Chem
698. Umezaki Y, Yasuyama K, Nakagoshi H, Tomioka K (2011) Blocking synaptic transmission with
tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian
clock neurons of Drosophila melanogaster. J Insect Physiol 57 (9):1290-1299
699. Umezaki Y, Yoshii T, Kawaguchi T, Helfrich-Förster C, Tomioka K (2012) Pigment-dispersing
factor is involved in age-dependent rhythm changes in Drosophila melanogaster. J Biol
Rhythms 27 (6):423-432
700. Uryu O, Tomioka K (2010) Circadian oscillations outside the optic lobe in the cricket Gryllus
bimaculatus. J Insect Physiol 56 (9):1284-1290
701. Usdin TB, Bonner TI, Mezey E (1994) Two receptors for vasoactive intestinal polypeptide with
similar specificity and complementary distributions. Endocrinology 135 (6):2662-2680
702. Ushirogawa H, Abe Y, Tomioka K (1997) Circadian locomotor rhythms in the cricket, Gryllodes
sigillatus. II. Interactions between bilaterally paired circadian pacemakers. Zoolog Sci 14
(5):729-736
703. van Corven EJ, Groenink A, Jalink K, Eichholtz T, Moolenaar WH (1989) Lysophosphatidateinduced cell proliferation: identification and dissection of signaling pathways mediated by G
proteins. Cell 59 (1):45-54
704. van den Berg MJ, Ziegelberger G (1991) On the function of the pheromone binding protein in
the olfactory hairs of Antheraea polyphemus. Journal of Insect Physiology 37 (1):79-85
705. Vanecek J, Watanabe K (1998) Melatonin inhibits the increase of cyclic AMP in rat
suprachiasmatic neurons induced by vasoactive intestinal peptide. Neurosci Lett 252 (1):2124
706. Vecsey CG, Pirez N, Griffith LC (2013) The Drosophila neuropeptides PDF and sNPF have
opposing electrophysiological and molecular effects on central neurons. J Neurophysiol
707. Veenstra JA, Agricola HJ, Sellami A (2008) Regulatory peptides in fruit fly midgut. Cell Tissue Res
334 (3):499-516
708. Veleri S, Brandes C, Helfrich-Förster C, Hall JC, Stanewsky R (2003) A self-sustaining, lightentrainable circadian oscillator in the Drosophila brain. Curr Biol 13 (20):1758-1767
709. Venthur H, Mutis A, Zhou J-J, Quiroz A (2014) Ligand binding and homology modelling of insect
odorant-binding proteins. Physiological Entomology 39 (3):183-198
710. Viana F, Van den Bosch L, Missiaen L, Vandenberghe W, Droogmans G, Nilius B, Robberecht W
(1997) Mibefradil (Ro 40-5967) blocks multiple types of voltage-gated calcium channels in
cultured rat spinal motoneurones. Cell Calcium 22 (4):299-311
711. Vickers NJ (2006) Winging it: moth flight behavior and responses of olfactory neurons are
shaped by pheromone plume dynamics. Chem Senses 31 (2):155-166
712. Villet RH (1978) Mechanism of insect sex-pheromone sensory transduction: Role of adenyl
cyclase. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology 61
(2):389-394
713. Vogt RG, Riddiford LM (1981) Pheromone binding and inactivation by moth antennae. Nature
293 (5828):161-163
714. Vogt RG, Miller NE, Litvack R, Fandino RA, Sparks J, Staples J, Friedman R, Dickens JC (2009) The
insect SNMP gene family. Insect Biochem Mol Biol 39 (7):448-456
715. Völkner M, Lenz-Bohme B, Betz H, Schmitt B (2000) Novel CNS glutamate receptor subunit
genes of Drosophila melanogaster. J Neurochem 75 (5):1791-1799
716. von Frisch K (1921) Über den Sitz des Geruchssinnes bei Insekten. Zool Jahrb Abt Allgem Zool
Physiol Tiere 38:449-516
717. Vosko AM, Schroeder A, Loh DH, Colwell CS (2007) Vasoactive intestinal peptide and the
mammalian circadian system. Gen Comp Endocrinol 152 (2-3):165-175
718. Vosshall LB (2003) Diversity and expression of odorant receptors in Drosophila. In: Blomquist G,
Vogt RG (eds) Insect Pheromone Biochemistry and Molecular Biology. Elsevier Academic
Press., pp 567-591
274
Bibliography
719. Vosshall LB, Hansson BS (2011) A unified nomenclature system for the insect olfactory
coreceptor. Chem Senses 36 (6):497-498
720. Vosshall LB, Wong AM, Axel R (2000) An olfactory sensory map in the fly brain. Cell 102 (2):147159
721. Vosshall LB, Amrein H, Morozov PS, Rzhetsky A, Axel R (1999) A spatial map of olfactory receptor
expression in the Drosophila antenna. Cell 96 (5):725-736
722. Wang X, Zhong M, Wen J, Cai J, Jiang H, Liu Y, Aly SM, Xiong F (2012) Molecular characterization
and expression pattern of an odorant receptor from the myiasis-causing blowfly, Lucilia
sericata (Diptera: Calliphoridae). Parasitol Res 110 (2):843-851
723. Wang YY, Aghajanian GK (1989) Excitation of locus coeruleus neurons by vasoactive intestinal
peptide: evidence for a G-protein-mediated inward current. Brain Res 500 (1-2):107-118
724. Wang YY, Aghajanian GK (1990) Excitation of locus coeruleus neurons by vasoactive intestinal
peptide: role of a cAMP and protein kinase A. J Neurosci 10 (10):3335-3343
725. Wanner KW, Nichols AS, Allen JE, Bunger PL, Garczynski SF, Linn CE, Robertson HM, Luetje CW
(2010) Sex pheromone receptor specificity in the European corn borer moth, Ostrinia
nubilalis. PLoS One 5 (1):e8685
726. Wegener JW, Hanke W, Breer H (1997) Second messenger-controlled membrane conductance in
locust (Locusta migratoria) olfactory neurons. J Insect Physiol 43 (6):595-603
727. Wei H (2012) Photoperiod-dependent plasticity of circadian pacemaker center in the brain of
the Madeira cockroach Rhyparobia maderae. PhD thesis, University of Kassel, Kassel
728. Wei H, Stengl M (2011) Light affects the branching pattern of peptidergic circadian pacemaker
neurons in the brain of the cockroach Leucophaea maderae. J Biol Rhythms 26 (6):507-517
729. Wei H, Stengl M (2012) Ca2+ -dependent ion channels underlying spontaneous activity in insect
circadian pacemaker neurons. Eur J Neurosci 36 (8):3021-3029
730. Wei H, el Jundi B, Homberg U, Stengl M (2010) Implementation of pigment-dispersing factorimmunoreactive neurons in a standardized atlas of the brain of the cockroach Leucophaea
maderae. J Comp Neurol 518 (20):4113-4133
731. Wei H, Yasar H, Funk NW, Giese M, Baz el S, Stengl M (2014) Signaling of pigment-dispersing
factor (PDF) in the Madeira cockroach Rhyparobia maderae. PLoS One 9 (9):e108757
732. Welsh DK, Logothetis DE, Meister M, Reppert SM (1995) Individual neurons dissociated from rat
suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14
(4):697-706
733. Werckenthin A (2013) Characterization of the molecular clockwork in the cockroach Rhyparobia
maderae - The core feedback loop genes period, timeless 1 and cryptochrome 2. PhD Thesis,
University of Kassel, Kassel
734. Werckenthin A, Derst C, Stengl M (2012) Sequence and Expression of per, tim1, and cry2 Genes
in the Madeira Cockroach Rhyparobia maderae. J Biol Rhythms 27 (6):453-466
735. Wetzel CH, Behrendt HJ, Gisselmann G, Stortkuhl KF, Hovemann B, Hatt H (2001) Functional
expression and characterization of a Drosophila odorant receptor in a heterologous cell
system. Proc Natl Acad Sci U S A 98 (16):9377-9380
736. Wever EG, Bray CW (1930) Present possibilities for auditory theory. Psychol Rev 37:365-380
737. Wever EG, Bray CW (1937) The perception of low tones and the resonance-volley theory. The
Journal of Psychology 3 (1):101-114
738. Wheeler DA, Hamblen-Coyle MJ, Dushay MS, Hall JC (1993) Behavior in light-dark cycles of
Drosophila mutants that are arrhythmic, blind, or both. J Biol Rhythms 8 (1):67-94
739. Wicher D (2001) Peptidergic modulation of insect voltage-gated Ca2+ currents: role of resting
Ca2+ current and protein kinases A and C. J Neurophysiol 86 (5):2353-2362
740. Wicher D (2015) Olfactory signaling in insects. Prog Mol Biol Transl Sci 130:37-54
741. Wicher D, Walther C, Penzlin H (1994) Neurohormone D induces ionic current changes in
cockroach central neurones. J Comp Physiol A 174 (4):507-515
275
Bibliography
742. Wicher D, Walther C, Wicher C (2001) Non-synaptic ion channels in insects - basic properties of
currents and their modulation in neurons and skeletal muscles. Prog Neurobiol 64 (5):431525
743. Wicher D, Schäfer R, Bauernfeind R, Stensmyr MC, Heller R, Heinemann SH, Hansson BS (2008)
Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation
channels. Nature 452 (7190):1007-1011
744. Wicher D, Agricola HJ, Sohler S, Gundel M, Heinemann SH, Wollweber L, Stengl M, Derst C
(2006) Differential receptor activation by cockroach adipokinetic hormones produces
differential effects on ion currents, neuronal activity, and locomotion. J Neurophysiol 95
(4):2314-2325
745. Wiedenmann G (1980) Two peaks in the activity rhythm of cockroaches controlled by one
circadian pacemaker. J Comp Physiol 137 (3):249-254
746. Wiedenmann G (1983) Splitting in a circadian activity rhythm: The expression of bilaterally
paired oscillators. J Comp Physiol 150 (1):51-60
747. Wiedenmann G (1984) Zeitgeber and circadian activity rhythms of cockroaches: A system of
unipolar coupled oscillators. Journal of Interdisiplinary Cycle Research 15 (1):69-80
748. Wiedenmann G, Lukat R, Weber F (1986) Cyclic layer deposition in the cockroach endocuticle: A
circadian rhythm? Journal of Insect Physiology 32 (12):1019-1027
749. Williams JA, Su HS, Bernards A, Field J, Sehgal A (2001) A circadian output in Drosophila
mediated by neurofibromatosis-1 and Ras/MAPK. Science 293 (5538):2251-2256
750. Wistrand M, Kall L, Sonnhammer EL (2006) A general model of G protein-coupled receptor
sequences and its application to detect remote homologs. Protein Sci 15 (3):509-521
751. Worster AS, Seabrook WD (1989) Electrophysiological investigation of diel variations in the
antennal sensitivity of the male spruce budworm moth Choristoneura fumiferana
(Lepidoptera: Tortricidae). Journal of Insect Physiology 35 (1):1-5
752. Wu L, Bauer CS, Zhen XG, Xie C, Yang J (2002) Dual regulation of voltage-gated calcium channels
by PtdIns(4,5)P2. Nature 419 (6910):947-952
753. Wu Y, Cao G, Nitabach MN (2008a) Electrical silencing of PDF neurons advances the phase of
non-PDF clock neurons in Drosophila. J Biol Rhythms 23 (2):117-128
754. Wu Y, Cao G, Pavlicek B, Luo X, Nitabach MN (2008b) Phase coupling of a circadian neuropeptide
with rest/activity rhythms detected using a membrane-tethered spider toxin. PLoS Biol 6
(11):e273
755. Wu ZN, Chen X, Du YJ, Zhou JJ, Zhuge QC (2013) Molecular identification and characterization of
the Orco orthologue of Spodoptera litura. Insect Sci 20 (2):175-182
756. Wülbeck C, Grieshaber E, Helfrich-Förster C (2008) Pigment-dispersing factor (PDF) has different
effects on Drosophila's circadian clocks in the accessory medulla and in the dorsal brain. J
Biol Rhythms 23 (5):409-424
757. Xia J, Ren D (2009) The BSA-induced Ca2+ influx during sperm capacitation is CATSPER channeldependent. Reprod Biol Endocrinol 7:119
758. Xia Y, Zwiebel LJ (2006) Identification and characterization of an odorant receptor from the
West Nile virus mosquito, Culex quinquefasciatus. Insect Biochem Mol Biol 36 (3):169-176
759. Xia Y, Wang G, Buscariollo D, Pitts RJ, Wenger H, Zwiebel LJ (2008) The molecular and cellular
basis of olfactory-driven behavior in Anopheles gambiae larvae. Proc Natl Acad Sci U S A 105
(17):6433-6438
760. Xu P, Garczynski SF, Atungulu E, Syed Z, Choo YM, Vidal DM, Zitelli CH, Leal WS (2012) Moth sex
pheromone receptors and deceitful parapheromones. PLoS One 7 (7):e41653
761. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M,
Tei H (2000) Resetting central and peripheral circadian oscillators in transgenic rats. Science
288 (5466):682-685
762. Yang Y, Krieger J, Zhang L, Breer H (2012) The olfactory co-receptor Orco from the migratory
locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and
expression pattern. Int J Biol Sci 8 (2):159-170
276
Bibliography
763. Yang Z, Sehgal A (2001) Role of molecular oscillations in generating behavioral rhythms in
Drosophila. Neuron 29 (2):453-467
764. Yao CA, Carlson JR (2010) Role of G-proteins in odor-sensing and CO2-sensing neurons in
Drosophila. J Neurosci 30 (13):4562-4572
765. Yao CA, Ignell R, Carlson JR (2005) Chemosensory coding by neurons in the coeloconic sensilla of
the Drosophila antenna. J Neurosci 25 (37):8359-8367
766. Yasar H (2013) Patch-Clamp-Analyse der PDF-Reaktionen circadianer Schrittmacherneurone der
Schabe Rhyparobia maderae. Diploma thesis, University of Kassel, Kassel
767. Yasuyama K, Meinertzhagen IA (2010) Synaptic connections of PDF-immunoreactive lateral
neurons projecting to the dorsal protocerebrum of Drosophila melanogaster. J Comp Neurol
518 (3):292-304
768. Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo
OJ, Menaker M, Takahashi JS (2004) PERIOD2::LUCIFERASE real-time reporting of circadian
dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad
Sci U S A 101 (15):5339-5346
769. Yoshii T, Rieger D, Helfrich-Forster C (2012) Two clocks in the brain: an update of the morning
and evening oscillator model in Drosophila. Prog Brain Res 199:59-82
770. Yoshii T, Todo T, Wulbeck C, Stanewsky R, Helfrich-Forster C (2008) Cryptochrome is present in
the compound eyes and a subset of Drosophila's clock neurons. J Comp Neurol 508 (6):952966
771. Yoshii T, Heshiki Y, Ibuki-Ishibashi T, Matsumoto A, Tanimura T, Tomioka K (2005) Temperature
cycles drive Drosophila circadian oscillation in constant light that otherwise induces
behavioural arrhythmicity. Eur J Neurosci 22 (5):1176-1184
772. Yoshii T, Wulbeck C, Sehadova H, Veleri S, Bichler D, Stanewsky R, Helfrich-Förster C (2009) The
neuropeptide pigment-dispersing factor adjusts period and phase of Drosophila's clock. J
Neurosci 29 (8):2597-2610
773. Young ED, Robert JM, Shofner WP (1988) Regularity and latency of units in ventral cochlear
nucleus: implications for unit classification and generation of response properties, vol 60. vol
1.
774. Yu Q, Jacquier AC, Citri Y, Hamblen M, Hall JC, Rosbash M (1987) Molecular mapping of point
mutations in the period gene that stop or speed up biological clocks in Drosophila
melanogaster. Proc Natl Acad Sci U S A 84 (3):784-788
775. Yu W, Zheng H, Houl JH, Dauwalder B, Hardin PE (2006) PER-dependent rhythms in CLK
phosphorylation and E-box binding regulate circadian transcription. Genes Dev 20 (6):723733
776. Zagranichnaya TK, Wu X, Villereal ML (2005) Endogenous TRPC1, TRPC3, and TRPC7 proteins
combine to form native store-operated channels in HEK-293 cells. J Biol Chem 280
(33):29559-29569
777. Zeng H, Qian Z, Myers MP, Rosbash M (1996) A light-entrainment mechanism for the Drosophila
circadian clock. Nature 380 (6570):129-135
778. Zerr DM, Hall JC, Rosbash M, Siwicki KK (1990) Circadian fluctuations of period protein
immunoreactivity in the CNS and the visual system of Drosophila. J Neurosci 10 (8):27492762
779. Zhang HJ, Anderson AR, Trowell SC, Luo AR, Xiang ZH, Xia QY (2011) Topological and functional
characterization of an insect gustatory receptor. PLoS One 6 (8):e24111
780. Zhao H, Gao P, Zhang C, Ma W, Jiang Y (2013) Molecular identification and expressive
characterization of an olfactory co-receptor gene in the Asian honeybee, Apis cerana cerana.
J Insect Sci 13:80
781. Zhao X, Salgado VL, Yeh JZ, Narahashi T (2003) Differential actions of fipronil and dieldrin
insecticides on GABA-gated chloride channels in cockroach neurons. J Pharmacol Exp Ther
306 (3):914-924
277
Bibliography
782. Zhao X, Salgado VL, Yeh JZ, Narahashi T (2004) Kinetic and pharmacological characterization of
desensitizing and non-desensitizing glutamate-gated chloride channels in cockroach neurons.
Neurotoxicology 25 (6):967-980
783. Zhao X, Yeh JZ, Salgado VL, Narahashi T (2005) Sulfone metabolite of fipronil blocks aminobutyric acid- and glutamate-activated chloride channels in mammalian and insect
neurons. J Pharmacol Exp Ther 314 (1):363-373
784. Zheng W, Zhu C, Peng T, Zhang H (2012) Odorant receptor co-receptor Orco is upregulated by
methyl eugenol in male Bactrocera dorsalis (Diptera: Tephritidae). J Insect Physiol 58
(8):1122-1127
785. Zheng X, Koh K, Sowcik M, Smith CJ, Chen D, Wu MN, Sehgal A (2009) An isoform-specific
mutant reveals a role of PDP1ε in the circadian oscillator. J Neurosci 29 (35):10920-10927
786. Zhou X, Yuan C, Guo A (2005) Drosophila olfactory response rhythms require clock genes but not
pigment dispersing factor or lateral neurons. J Biol Rhythms 20 (3):237-244
787. Zhou YL, Zhu XQ, Gu SH, Cui HH, Guo YY, Zhou JJ, Zhang YJ (2013) Silencing in Apolygus lucorum
of the olfactory coreceptor Orco gene by RNA interference induces EAG response declining
to two putative semiochemicals. J Insect Physiol
788. Zhu H, Yuan Q, Briscoe AD, Froy O, Casselman A, Reppert SM (2005) The two CRYs of the
butterfly. Curr Biol 15 (23):R953-954
789. Zhu Y, Yakel JL (1997) Modulation of Ca2+ currents by various G protein-coupled receptors in
sympathetic neurons of male rat pelvic ganglia. J Neurophysiol 78 (2):780-789
790. Ziegelberger G, van den Berg MJ, Kaissling KE, Klumpp S, Schultz JE (1990) Cyclic GMP levels and
guanylate cyclase activity in pheromone-sensitive antennae of the silkmoths Antheraea
polyphemus and Bombyx mori. J Neurosci 10 (4):1217-1225
791. Zufall F, Hatt H (1991a) Dual activation of a sex pheromone-dependent ion channel from insect
olfactory dendrites by protein kinase C activators and cyclic GMP. Proc Natl Acad Sci U S A 88
(19):8520-8524
792. Zufall F, Hatt H (1991b) A calcium-activated nonspecific cation channel from olfactory receptor
neurones of the silkmoth Antheraea polyphemus. J Exp Biol 161:455-468
793. Zufall F, Stengl M, Franke C, Hildebrand JG, Hatt H (1991) Ionic currents of cultured olfactory
receptor neurons from antennae of male Manduca sexta. J Neurosci 11 (4):956-965
278
Acknowledgements
7 Acknowledgements
First of all I would like to thank Prof. Dr. Monika Stengl for giving me the opportunity for this thesis,
for her introduction into the interesting topics of insect chronobiology and olfaction, for many
encouraging discussions, for her patience, and her trust placed in me. Special thanks goes to
Dr. Dieter Wicher for supervising me, for many interesting conversations, and for reviewing this
thesis. I want to thank Prof. Dr. Bill Hansson for the opportunity to work in his amazing lab at the
Max Planck Institute for Chemical Ecology in Jena for almost two years.
I am very thankful to Hanzey Yasar, Janis Brusius, Latha Mukunda, Anastasia Pyanova, and Simone
Achenbach, who contributed to this work. Special thanks goes to Nils-Lasse Schneider, Keram
Pfeiffer, Steffi Krannich, Wolfgang Schwippert, Dieter Wicher, Veit Grabe, Marco Schubert, and
Antonia Strutz for introducing me into electrophysiology and calcium imaging. I want to thank Achim
Werckenthin for Matlab support, many helpful discussions, proofreading of the manuscript, and his
help in many situations. Additionally, I thank Robin Schumann for proofreading, Steffen Klingenhöfer
and Andreas Nolte for Matlab support, as well as Ewald Grosse-Wilde, Markus Knaden, Shannon
Olsson, Jürgen Rybak, Wolfgang Schwippert, and Richard Vogt for many helpful discussions. I want to
express my thanks to Bernhard Petri, Petra Schulte, Daniela Gocht, and Kathrin Göbbels for helpful
comments regarding primary cell cultures. I am also thankful to Sascha Bucks, Toni Burmeister, Sylke
Dietel-Gläßer, Karin Große-Mohr, Sabine Kaltofen, Gisela Kaschlaw, Swetlana Laubrich, Ursula
Reichert, Frank Richter, Christin Sender, Jutta Seyfarth, Regina Stieber, Silke Trautheim, Christa
Uthof, Daniel Veit, Kerstin Weniger, and Christina Wollenhaupt for their assistance. Furthermore, I
wish to thank all members of the labs in Marburg, Kassel, and Jena, whom I head the pleasure of
getting to know, for the good collaboration and the nice atmosphere. Special thanks goes to Marius
Bartholmai, the second Ronshäuser in the department, and my office mates (Ildefonso AtienzaLopez, Janis Brusius, El-Sayed Baz, Hany Dweck, Christian Flecke, Loreen van Heinrich, Christian
Klinner, Christopher König, Sarah Körte, Anna Kretschmer, Alexia Loste, Andreas Nolte, Kerstin
Pasemann, Wolfgang Schwippert, Achim Werckenthin, and Hanzey Yasar) and flat mates (Uli Ertelt,
Marcus Gaudigs, Torsten Hartung, Sandra Hitzel, Stephan Kleiner, Sophie Mohn, Anne Placke, Sarah
Schröder, and Anne Wenck) for their company and the great time.
Finally, I would particularly like to thank my sister and my parents for their patience, encouragement,
and support, without which the accomplishment of this thesis would not have been possible.
I do not want to thank the drummer, who thought it was a good idea to have his practice sessions in
the room below my office.
279

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